A Novel Stem Cell Delivery Device

Final Report

Project Sponsor: Dr. Luis Garza M.D., Ph.D., Department of Dermatology, Johns Hopkins Hospital

Design Team 8: Michael Clark (Team Leader), Angelica Herrera, Arianne Papa, Seung Jung, Michael Mow,Annabeth Rodriguez, Jose Solis, Prateek Gowda

Contents

1 Abstract 3

2 Introduction 42.1 Clinical Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.2 Clinical Need . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.3 Solution and Design Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3 Design 63.1 Design Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.2 Device Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.3 Base Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.4 Cell Thawing Subsystem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.5 Peristaltic Pump Subsystem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83.6 Closed-Loop Cell Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83.7 Cryobag Freezing Mold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83.8 Power Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93.9 Angle and Depth Guards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

4 Results 104.1 Post-Injection Cell Viability Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

4.1.1 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104.1.2 Testing and Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

4.2 Cell Thawing System Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Appendix A Materials 12

Appendix B Prototype Budget 13

Appendix C Figures, Photos, and Sketches 14C.1 Injector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14C.2 Base Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14C.3 Motor and Driver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15C.4 Onboard Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15C.5 Microcontroller . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15C.6 Thermocouple . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16C.7 Cryobag Freezing Mold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16C.8 Cryobag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16C.9 Luer Lock Connector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17C.10 Needle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17C.11 Power Delivery Schematic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17C.12 Microcontroller Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18C.13 Heating System Performance Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19C.14 Viability Testing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Appendix D Calculations 20D.1 Volume Resolution Calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20D.2 Tubing Dead Space Calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Appendix E References 21

Appendix F Proposal 22

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1 Abstract

Recent advancements in stem cell therapies have shown the potential to revolutionize the treatment of manyconditions. However, there is still a need to consistently and accurately deliver stem cells to target regionsin the solid organs of the body–particularly the skin. Skin stem cell therapies currently under investigationhave the potential to reverse hair loss, heal wounds, and alter the phenotype of the epidermis. We havedesigned a device to deliver stem cells to the skin at adjustable volumes with minimal viability loss or riskof contamination. The device makes use of a peristaltic pump to provide precise control over injection rate,volume, and the resulting shearing forces. Cell viability is also improved through an automated heatingsystem that thaws the cells at an expeditious but controlled rate immediately prior to injection. The entirecell pathway is a closed loop, which minimizes the risk of contamination. Cryobags were utilized to increasethe volume of cells available to the physician administering the therapy. Interchangeable angle and depthguards were designed to improve the consistency and repeatably of injections. In preparation for ex vivotesting, an exploratory in vitro test was performed on the injection mechanism using fibroblast cells. Thethermal performance of the heating system was also evaluated experimentally.

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2 Introduction

2.1 Clinical Problem

Stem cell therapies are an expanding market in regenerative medicine. The global stem cell industry hasgrown 13.6% annually from $5.6 billion in the year 2013 [1]. Currently, 4,681 clinical trials involving stemcell therapies are being carried out in the United States with over 3,000 in early phases of testing [2]. TheNIH estimates spending approximately $2.77 billion on stem cell research in 2015 [3]. Public funding fromindividual states will total over $4.1 billion by 2018 [4]. As thousands of therapies advance past initial stagesof testing, there will be an increased demand for a safe and effective way to deliver the stem cells to patients.

Current stem cell therapies for the skin involve altering the characteristics of dermal cells to treat wounds,rashes, and burns. In the United States, the treatment of wounds and associated complications exceeds $20billion annually [5]. Existing treatments for these conditions are prohibitively expensive; skin allografttherapies typically cost $500,000 per patient [6]. Skin stem cell therapies also have cosmetic applicationssuch as regenerating hair follicles, repairing scar tissue, and changing the phenotypic expression of the skin.

Today, physicians have difficulty delivering stem cells to the skin in a consistent manner. Providers relyon tactile feedback and previous experience to deliver the cells to the desired location. Subjectivity andvariance are inherent in the current treatment administration procedure. In order to generate useful clinicaltrial data, the stem cell delivery procedure needs to be optimized to be as consistent and repeatable aspossible.

A primary concern is ensuring post-injection cell viability. If cells are injected too quickly, shearingforces in the needle can tear them apart. Another concern is contamination: in the current procedure, cellsare exposed to air when they are transferred from the freezing vessel to the syringe. This can lead to cellcontamination and an increased risk of post-treatment infection. As a result of these complications, manyexisting dermal injectors cannot be adapted to this highly specialized application.

2.2 Clinical Need

No devices on the market are specifically designed to mitigate the difficulties associated with delivering stemcell therapies to the skin. Consequently, there is a need for a device that allows physicians to deliver stemcells to target dermal regions at adjustable volumes with minimal risk of contamination or viability loss.

2.3 Solution and Design Specifications

The team’s dermal injector addresses the biological hurdles associated with stem cell delivery by providingadjustable injection rates, integrated cell thawing capabilities, and a closed-loop delivery system that reducesthe risk of contamination. Stem cells are commonly stored and frozen in cryogenic vials, which typicallyhold a one to two milliliter volume. The team will make use of cryogenic bags, which can hold volumesranging from five to ten milliliters. This reduces the number of times the physician has to reload the device– effectively decreasing procedure time and air exposure, thus reducing the risk of contamination. Thecryobag will be connected to a cartridge-tubing system and a needle to form the closed-loop system. Thephysicians will place the cartridge onto a peristaltic pump, which will accurately output desired volumes withan accuracy of ±1 µl and physician-specified rates for each injection averaged at one minute per injection.

To further enhance cell viability, the team spoke with Dr. Luis Garza, M.D., Ph.D., who is the sponsorand medical adviser for the team, and found that a heating system should be included as well.

Several other possibilities why a new device might be better than a syringe might be the problemof temperature, where we know that cells will likely be shipped to a consumer frozen. Butfrozen cells lose viability or thaw too slowly. If they are thawed more quickly, then viability ismaintained better, so a device that eliminates user variability in terms of quick thawing ratecould also improve final outcome for cellular therapies.

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An integrated cell thawing system was designed that heats frozen stem cells at a consistent rate, whichdramatically improves cell viability [7]. In order to achieve this, the frozen stem cells must be heated from−196 C, the temperature of the liquid nitrogen, to 37 C, the average temperature of the human body, inunder two minutes per 1.0 ml thawed, which is the current standard established from a traditional waterbath.

The dermal injector will be kept in its base station when not in use. This docking station will chargethe device and will be used to adjust device settings. Every system in the dermal injector will be controlledelectronically. The physician will only have to input his or her desired injection volume and injection rate,and then start the thawing process using a touch screen user interface on the base station. This LCD touchscreen will indicate when cell thawing is complete and alert the physician when the device is ready to use.This simplified procedure is to ensure that this method is no more difficult than the current standard of care.

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3 Design

3.1 Design Process

The team conducted months of extensive research to gain a thorough understanding of the market. Anintellectual property search was conducted, which revealed ample room to innovate as there were no otherdevices that delivered stem cells to the dermis. This meant that the team had no competitors, but also hadno predicate devices upon which to improve their design. Therefore, the group decided to expand the searchto include devices that delivered stem cells to other organs of the body. However this also revealed a lackof devices and the team switched to drug delivery methods instead. As a result, the team developed fourpromising devices that could have been adapted to deliver stem cells.

The first idea was a direct stem cell insertion. This method was considered as it was the simplest wayto get stem cells from point A to point B. The stem cells would be cultured in a laboratory setting into acell matrix. The method would then require an operation where the patient’s skin was cut and pulled back,the cell matrix inserted into place, and then skin sutured back on. Although this method was the mostdirect treatment, it was also the most invasive. The team thought it best to avoid operations that requireda lengthy recovery time and continued to seek less invasive methods of delivery.

The next idea was an injection gun. This was by far the fastest way the team found to deliver a treatment.This device penetrated the skin with the aid of pressurized gas, which evenly distributed the drug. However,this method raised concerns as the velocity of injection was deemed detrimental to the viability of the cells.In addition, the location that the stem cells were delivered also raised concerns as the cells traveled too deepinto the dermis and passed its optimal location. From this idea, the team determined that a slow consistentrate of injection was a vital part of the device as it ensured that the cells maintained a <40% reduction inviability.

The use of a microneedle array was one of the best methods to deliver drugs at the optimal location. Thisdevice consisted of a series of microneedles that were able to deliver drugs at consistent rates. However, theteam found that a small needle gauge would damage the cells and lower cell viability as it passed throughthe needle. Though this method had a consistent flow rate, the team determined that the correct needlegauge was just as important. After an extensive literature search, the team pursued a 23 gauge needle,which was found to be the most optimal size for their objective as it was big enough for the cells to travelthe needle without much shearing, but small enough to penetrate the skin without the pain associated withbigger needles.8

The final idea was an insulin pen injector. Similar to a needle and syringe, this device simplified thedelivery process. The patient would set the volume he or she needed and then press a button to deliver thetreatment. Using this method as a springboard, the team adapted this simple design and incorporated allthe features that the team determined was vital from the previous ideas: minimal invasiveness, consistentflow rate, use of a 23 gauge needle, and adjustable volume concentration from the pen injector.

From there, the team generated ideas and selected designs that yielded the best treatment outcomes andper-dollar performance. After soliciting feedback from experts, the team modified the device to include theelements described in §3.2.

3.2 Device Overview

The device is a hand-held electronic cell injection system (Appendix C.1). The injector itself is cordless– it docks in a powered base station, which allows the device to be charged and programmed by the user(Appendix C.2). The injector has four discrete subsystems: an automated cell thawing system with atemperature feedback mechanism, a peristaltic pump system that offers precise control over injection rateand volume, a closed-loop cell pathway, and a power delivery system. A sealed septum separates the electronicsystems from the “wet” cellular pathway, which effectively mitigates the risk of electrical shock in the caseof a breach in the closed-loop system.

The injector device is designed to be held like a computer mouse, with the pointer finger resting on theinjection button. This position provides a great deal of stability to the physician administering the therapy.

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The long tip of the device allows clinicians to achieve very shallow injection angles. The tip also acceptsinterchangeable angle and depth guards.

The base station houses an onboard computer (Raspberry Pi Model B+) and a touch-screen user interface.A bag freezing mold was also developed that facilitates increased heat transfer from the heating pads to thecryobag.

3.3 Base Station

The base station (Appendix C.2) was developed as a means of improving physician usability by utilizing alarge full color capacitive LCD touchscreen with a user-friendly graphic user interface (GUI), which allows thephysician to set the parameters of the injection easily. After the physician attaches the cell cryobag cartridgeto the device, the program switches on and prompts the physician to enter the desired injection volume ofstem cells After the physician inputs the information, the data is sent to an internal computer inside the basestation. The Raspberry Pi was utilized as the internal computer (Appendix C.4) due to its compatibility withtouch screen interfaces and its ability to communicate with our specific microcontroller, the Arduino Micro(Appendix C.5). The internal computer carries out calculations from the selected volume of injection inorder to determine the time necessary for cell thawing, while automatically setting the injection rate. Insidethe handheld device, The data computed by the internal computer is programmed onto the microcontrollerin the device via contacts between the base station and the resting device. The Arduino carries out theheating process and signals when the process is complete. The screen then prompts the physician to removethe device from the base station and begin the injection.

3.4 Cell Thawing Subsystem

The heating system functions to thaw the stem cells quickly, but in a controlled manner that prevents therisk of destroying the cells due to overheating. Thawing cells quickly helps reduce the risk that the cellswill be damaged by ice crystals that are present during the thawing process. Literature also suggests thatrapid thawing reduces protein damage [8]. The heating process takes place while the device is resting in thebase station. The physician places the cryobag cartridge into the device and selects the heating option onthe base station user interface. The cells are thawed by two heating pads that envelope the cryobag. Dueto the large surface area of the cryobag and thin distribution of the cells in the bag itself, the heating isuniform along the cells in the bag. The heating pads maintain a constant temperature of 37.0 C. This isthe ideal temperature of the cells inside the human body. It is important the heating pads do not exceedthis temperature, as it leads to potential cell death and an overall loss in cell viability. To achieve constanttemperature control, the system utilizes a negative feedback loop and control systems to provide safe heatingof the cells.

The heating subsystem is comprised of two thin parallel heating pads constructed using a mesh of polyesterfilament and conductive fiber folded into a protective Polyimide Film. The fact that these are low power,flexible and draw little power makes them ideal for things like hand-warmers and other heated garments. Thecurrent source for these heating pads is controlled and obtained from a regulated DC power supply providedby a grounded wall outlet. The circuit is regulated by an NPN transistor switch that can be opened and closedbased on feedback from data obtained by a thermocouple (Appendix C.6). The thermocouple concurrentlysends the temperature of the heating pads as digital data to an Arduino Micro (Appendix C.5) that thenregulates the switching of the transistor (See code in Appendix C.12). This transistor was used as a switchingmechanism over other options, such as mechanical relays for example, because of its solid state design. Thisallows the team to utilize power with modulation techniques to rapidly switch the current to more safelycontrol the heating of the pads. Using this method, the pads will heat rapidly at lower temperatures andslowly as the temperature approaches the maximum of 37.0 C. Once the cells have thawed, the heating hasbeen completed, at which time the physician will be signaled to remove the device from the base station andprepare for injection.

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3.5 Peristaltic Pump Subsystem

The pump system is designed to control the stem cell injection rates and reduce viability losses due toshearing forces in the needle. The user interface for the pumping process is comprised of two mechanicalbuttons on the device, a clear-air button and an injection button. The purpose of the clear-air button isto remove excess air in the tubing and replace it with the cell from the cryobag. This is performed at thediscretion of the physician and is analogous to clearing air bubbles in the common needle and syringe. Theclear-air function pumps at a higher rate than the injection button. The injection button, when toggledby the physician, pumps the stem cell solution out at a slow, constant rate to maximize cell viability andreduce shearing forces. Testing will be carried out to determine the ideal rate of injection. To perform theinjection, the physician inserts the needle into the patient and holds the injection button.

The pump system is comprised of a peristaltic pump head that is rotated by a small four-wire unipolarstepper motor (Appendix C.3). Peristaltic pumps work by pushing and collapsing the walls of a flexibletubing material to create a vacuum and a source of suction, that when rotating along the tubing walls,draws out the cells from the cryobag into the tubing. This method prevents contact between the cellsand any external pumping mechanism, preventing contamination during the injection. A stepper motor isutilized to turn the peristaltic pump and control the rotation without using a separate mechanical or opticalencoder. Removing the encoder prevents bulky attachments, reduces the weight and controls space withinthe device. Instead, stepper motors work by moving in steps that are designated by the Arduino Micro,which eliminates a need for a negative feedback loop.The motor is geared to afford a resolution of 1024 turnsper revolution, which gives a pumping resolution of 0.0455 µl based on the size of the tubing and pump headradius (Appendix D.1). This high resolution and controlled pump system ensures that even small volumesof stem cell solution can be injected without the device.

3.6 Closed-Loop Cell Pathway

The closed-loop cell pathway is comprised of four components: the cryobag, tubing, needle, and Luer-lockconnectors. Cryobags were chosen over more traditional cryovials because they are able to contain a larger(and more clinically appropriate) volume of cells - this decreases the frequency with which the physician hasto halt the procedure to load more cells into the device. OriGen Biomedicals PermaLife Cell Culture Bag(Appendix C.8) was selected for the device. The bag is made from biologically-inert Fluorinated ethylenepropylene (FEP), which offers an operational temperature range of −196 C to 137 C permitting the bag tobe frozen in liquid nitrogen and sterilized by autoclave.

Unlike the cryobag, the tubing will not be subjected to extreme temperatures. Clear silicone tubingmade from FDA-compliant resins (McMaster-Carr 5236K501) was selected for its plyability and ability tobe sterilized by autoclave. The inner diameter (ID) and length of the tubing was minimized (794 µm) inorder to reduce dead space losses, i.e. the volume of cells required to fill the tubing that become unavailablefor injection. In the current design, less than 76 µl of dead space exists in the tubing (Appendix D.2).

Hypodermic needles are widely available in health care settings, so the needle selection was guided byindustry standards. Becton, Dickinson, and Company (BD) has a significant share of the hypodermic needlemarket and is therefore a suitable supplier, although a wide variety of manufactures sell needles that can beused with the device. The device will accept any needle with a Luer-lock connector - various gauges, lengths,and bevels are available to suit a variety of procedures. A 23 gauge needle (Appendix C.10) was utilized fortesting purposes since it is commonly used in intradermal injection procedures.

All interfaces between the cryobag, tubing, and needle feature a Luer-lock type connector (AppendixC.9). Luer-lock is a commonly used and ISO-standard fitting that will be familiar to physicians. Luer-lockconnectors were chosen over Luer-slip connectors due to their ability to withstand higher injection pressures.

3.7 Cryobag Freezing Mold

Maximum heat transfer is achieved when the contact area between the heating pad and cryobag is maxi-mized. In order to increase the contact area, freezing the cryobag at a uniform (flat) thickness works to

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the physician’s advantage. A cryobag freezing mold (Appendix C.7, isometric drawing) was developed as ameans to that end. The mold allows two 10 ml cryobags to be frozen flat inside of a standard 135 mm by135 mm cryobox. Polylactide (PLA) was used to prototype the mold, but the final product will implement alow-temperature polymer capable of thermocycling from room temperature to liquid nitrogen temperatures(e.g. polypropylene). The cryobag sits in between the two halves of the mold. Stainless steel springs will beemployed to apply pressure on both sides, gently compressing the bag in the middle.

3.8 Power Systems

Two systems provide power to the device subsystems (Appendix C.11). A power supply in the base stationtransforms, rectifies, filters and regulates 120V AC current to 5.1V DC at 2.1A. The 5.1V rail is used topower the onboard computer in the base station, charge the 12V battery in the injector, and operate a NPNtransistor along with the Arduino. The power supply is controlled by a switch located on the back of thebase station. The second power source is the 12V battery in the injector, which allows the device to beoperated wirelessly when undocked from the base station. The battery supplies the power necessary to runthe onboard microcontroller that regulates the pump motor and thermocouple feedback system.

3.9 Angle and Depth Guards

A selection of nine angle and depth guards were designed. The guards clip on to the tip of the device.Three clinically relevant angles (90, 45, and 15) and three physiologically relevant depths (intradermal,subdermal, and intramuscular) were represented. The guards are made from clear plastic so that physicianscan visualize the injection site while they administer the cellular therapy.

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4 Results

4.1 Post-Injection Cell Viability Testing

4.1.1 Methods

After finalizing each component of the stem cell injector, preliminary testing for the two major subsystemsof the device was performed. Both the closed-loop cell injection system and automated cell thawing system(and feedback mechanism) were examined. Literature suggests that cell death occurs when cells are exposedto high levels of pressure or shear force, specifically above 1.0 Pascal [9]. It has been shown that viabilitylosses up to forty percent can occur when cells are injected through a needle (size) and syringe(rate, celldensity, media (PBS in this case)) due to shearing forces [10].

Preliminary tests were designed to correlate post-injection cell viability to injection rate through a 23gauge needle. A 23 gauge needle was chosen since this is the most commonly used needle size for cellviability testing and has clinical relevance. A needle of this size is optimal for the balance between a lowpain level for the patient and a large enough width to maintain cell viability [11]. Also, a small diameterallows precise regions, such as the dermoepidermal junction, to be reached. Injection rates of 6.0 mL/min,3.0 mL/min, 1.0 mL/min and 0.5 mL/min were chosen based on physician feedback and literature13. Thefastest injection rate (6.0 mL/min) was used to simulate high shear forces through the needle. An injectionrate of 3.0 mL/min mimicked a physicians typical injection speed. Slower injection rates (1.0 mL/min) wereused in other viability studies and used to simulate the lowest amount of shear forces through the needle(0.5mL/min).13

4.1.2 Testing and Results

Initial viability testing was performed with mouse spleen cells due to their relative availability. Cells wereobtained from Dr. Luis Garzas laboratory at Johns Hopkins Hospital.

Mouse spleen cells were harvested, filtered with PBS through a mesh netting to isolate the cells, andcentrifuged at 1000 RPM for 5 minutes. The cells were then resuspended in Phosphate-buffered saline(PBS). In order to determine cell density, 10 uL of the cell solution was mixed with 10 uL of trypan blueand placed on a hemocytometer slide. Using a Countess Automated Cell Counter, the number of live cellswas approximated. However, the countess did not provide clear estimates for cell density. This could be dueto the fact that red blood cells were not separated from the spleen cells. In future tests, the red blood cellscan be lysed and then washed away using ACK Lysing Buffer (Life Technologies, A10492-01).

Additional testing was performed with volar fibroblasts biopsied from a patients sole. Fibroblasts wereacquired from Dr. Luis Garzas laboratory at Johns Hopkins Hospital (IRB NA 00068684) and culturedin Dulbecco’s Modified Eagle Medium (DMEM) for one week before experimentation. Due to the shortexpansion time, cells were only used at a density of 3.0× 105 cells/mL.

Injection testing was performed using a 23 gauge needle and syringe. For the 6.0 ml/min rate, the injectionwas performed manually to model the accuracy and repeatability of a physician. For the slower rates (3.0,1.0, and 0.5 ml/min) a syringe infusion pump was used to provide consistent injection rates. In each trial,300 µl of the cell solution were loaded into the 1.0 ml syringe by hand. Using the infusion pump (or by handin the 6.0 ml/min trial), 100 µl of the solution were injected into a 1.0 ml vial at a predetermined controlledrate. The ejected cells were then placed on ice. Three trials were conducted for every injection rate. Toserve as a control, 100 µl of cell solution were drawn up manually into the syringe and injected into a 1.0 mlwithout using a needle. After completing all injections, 10 µl from each 1.0 ml vial were drawn up with apipette and placed in a new 1.0 mL vial with 10 µl of trypan blue. After mixing, 10 µl of the solution werepipetted onto a hemocytometer slide and imaged.

No losses in viability were seen during this experiment; all visible cells appeared viable with intactmembranes and no dark stains from the trypan blue (Appendix C.14). However, there was much variationbetween cell densities in each hemocytometer image. Across different injection rates, there did not seem to bea constantly increasing or decreasing trend in the number of cells present. Additionally, between injectionsof the the same rate, results would vary from no visible cells to large inordinate amounts of cells. As a

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result, standard deviation values from these injection rates tend to be greater than the average of observablecells within that rate. For example, the trials for the 1.0 ml/min injection rate were: 0 cells visible, 1cell, and 61 cells visible. This results in an average of 20 cells visible in this injection rate and a standarddeviation of 35 cells. These high standard deviation numbers made any quantifying results inconclusive orat least statistically invalid. In the future, cell solutions will be mixed thoroughly before pipetting onto thehemocytometer slide to ensure homogeneity throughout the solution. A 24 hour time point will be used aswell to see if viability losses are seen over time and not immediately after injection. By using trypan blue,cell death will only be seen once the cell membrane is lysed. This may not occur immediately since cellsmay only initially be damaged.

Revised testing is currently underway at Johns Hopkins University Homewood campus. Mouse fibrob-last (L929, P3 11334) cells were acquired from Dr. Elizabeth Logsdon. The cells, which were originallyfrozen at ninety percent confluence were thawed quickly and cultured in DMEM (10% FBS with Penicillin-Streptomycin (Sigma-Aldrich P4333, 5:500) for four days prior to testing.

Before experimentation, cell density was determined using standard cell counting procedures and a hemo-cytometer slide. Dimethyl sulfoxide (DMSO) was added to the cell solution to mimic the conditions in Dr.Garzas clinical trial. It is expected that DMSO will make the cells more susceptible to shearing forces andviability losses [12]. Many studies have demonstrated reduced cell viability in a dose-dependent manner.Although DMSO is used to protect cells while frozen, they may induce some cytotoxicity when they arethawed due to permeability [13]. The previous procedure using fibroblasts was slightly modified for thisexperimentation. Injections were performed directly into 24 well plates for easier imaging. An identicalsecond well plate was used for the 24 hour time point. Injections were repeated three times, either by handor with the infusion pump, per injection rate. Trypan blue was added to the wells before imaging.

4.2 Cell Thawing System Testing

The automated thawing system and feedback loop were thoroughly tested and examined. Before use in theclinical setting, stem cells are frozen in liquid nitrogen and stored. When needed, the physician removes andthaws these vials before injection. Cells can withstand a maximum temperature of 37 C, any higher andthey face the potential of cell death [14]. To maintain a consistent thawing procedure, the in device heatingcapabilities were examined. Testing the heating component of the dermal injector was vital to the device.

To ensure a maximum temperature of 37 C, the heating pads were connected to a thermocouple, recordingthe temperature during the experiment. Along the heating circuit, these pads were controlled by a switchingsystem and power supply. The thermocouple created a negative feedback loop, allowing us to reach amaximum temperature of 37 C. The arduino read the temperature from the thermocouple and controlledthe switch along the heating circuit.

The test was carried out with a supply voltage of 1.1V to the collector input of the transistor. Thefeedback program on the microprocessor utilized power width modulation techniques in three stages toregulate current flow to the heating pads. Rapid heating occurred when the temperature of heating padswere below 30 C. The temperature then increased slowly between the period of 30 C-37 C. Once the padshad reached 37 C, it began to oscillate along this target temperature, with maximum at 37.6 C (AppendixC.13).

The thermocouple feedback system, along with the PWM technique used in the programming, demon-strated the ability to control temperature with 1.0 C precision from the 37 C target temperature. Im-provements can be made in the software by increasing the reading rate of the program. In this test, thetemperature was taken at a rate of one reading every 250 milliseconds. Increasing this rate would increasethe number of times that the temperature is managed, resulting in smaller fluctuations along the targettemperature and a reduction in time between heating and cooling cycles.

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A Materials

System Component Description Source Item Number

Base Station Outer Shell PLA Makerbot Indus. White PLA

Computer Raspberry Pi Model B+ Raspberry Pi Model B+

Power Button SPST (Round) Sparkfun COM-11138

LCD Screen 2.8-Inch, TFT, 320x240 Adafruit Indus. 1601

Power Cord 18AWG, 72”, Black Digi-Key Elec. 221001-01

Contacts Universal 1.8MM SMD Digi-Key Elec. 1003-1010-2-ND

USB/Data Cable Adafruit 70

Device Outer Shell PLA Makerbot Indus. White PLA

Microcontroller Arduino Micro Arduino A000053

Buttons Tactile Button Assortment Sparkfun COM-10302

LEDs Blue LED Digi-Key Elec. 160-1602-ND

Pins Stainless Steel McMaster-Carr 6517K65

Springs Compression, Steel Conical McMaster-Carr 1692K11

Stepper Motor 12V Adafruit 918

Motor Driver Lighted Texas Instruments ULN2003ADR

Heating Pads Flexible Sparkfun COM-11289

Battery 12V Enegizer A23

Transistor General Purpose Transistor ON Semiconductor PN2222

Thermocouple K-type Adafruit Indus. 270

Transducer For K-type Thermocouple Adafruit Indus. 269

Resistors 10KΩ, 1KΩ Digi-Key Elec. CF14JT10K0,

CF14JT1K50

Cell Pathway Tubing For Peristaltic Pumps McMaster-Carr 5236K501

Needle 23 Gauge (for testing) Becton & Dickenson 305148

Cryobag 10 ml OriGen PL07

Leur Lock 0.8mm ID Barb Qosina 11106

Freezing Mold Mold PLA Makerbot Indus. Black PLA

Springs Conical, Stainless Steel McMaster-Carr 1692K12

Angle Guards Guard Clear, Polycarbonate McMaster-Carr 8585K51

12

B Prototype Budget

13

C Figures, Photos, and Sketches

C.1 Injector

C.2 Base Station

14

C.3 Motor and Driver

C.4 Onboard Computer

C.5 Microcontroller

15

C.6 Thermocouple

C.7 Cryobag Freezing Mold

C.8 Cryobag

16

C.9 Luer Lock Connector

C.10 Needle

C.11 Power Delivery Schematic

17

C.12 Microcontroller Code

18

C.13 Heating System Performance Data

C.14 Viability Testing Data

Figure 1: Cell viability testing using sole fibroblasts from Dr. Luis Garzas laboratory. Variability in celldensity is seen between experimental and control groups when injected onto a hemocytometer slide andimaged a) Injection rate of 1.0 mL per minute and b) control group injected by hand without a needle.

19

D Calculations

D.1 Volume Resolution Calculation

Stem cell therapies are often carried out with very small injection volumes, <10 ml. It is important, therefore,that the device is able to provide pumping resolutions small enough and with enough precision for any stemcell therapy that the physician might carry out. Volume resolution is characterized as the smallest volumethat the device can inject with one motion of the pump.

In our calculations, volume resolution was calculated as a function of the radius of the peristaltic pumphead, the step resolution of the peristaltic pump, and the interior diameter of the tubing.

The manufacturer’s specification sheet shows an interior tube diameter of 0.0794 cm, while the peristalticpump head is designed to be a half circle of a 1.5 cm radius. Therefore in one full rotation of the pump head,we calculate an output of 0.0466 ml of solution. The 1/64th geared stepper motor has a full 1024 steps perrotation.

Therefore there is a calculated volume resolution of 0.0455 µl.

D.2 Tubing Dead Space Calculation

As stated in Appendix D.1, the cross-sectional area of the tubing is:

A = π×r2 = π×(.0794

2

)2

= .0049514cm2

Approximately 15.24 cm of tubing is used in the device. The tubing dead space is therefore:

V = A×l = .0049514 ∗ 15.24 = .0754cm3

The total dead space is therefore 75.46 µl.

20

E References

[1] “Global Market for Stem Cells to Reach $10.6 Billion in 2018.” The Global Market for Stem Cells. BCCResearch, July 2014. Web. 19 Jan. 2015.[2] “Search Results.” Search Of: Stem+cell. ClinicalTrials.gov, n.d. Web. 19 Jan. 2015.[3] “Estimates of Funding for Various Research, Condition, and Disease Categories (RCDC).” NIH Cate-

gorical Spending. NIH, 7 Mar. 2014. Web. 19 Jan. 2015.[4] “Embryonic Stem Cell Research by the Numbers.” Center for American Progress, 17 Apr. 2007. Web.

19 Jan. 2015.[5] Chen, Ming, Melissa Przyborowski, and Francois Berthiaume. Stem Cells for Skin Tissue Engineering

and Wound Healing. Critical reviews in biomedical engineering 37.4-5 (2009): 399-421. Print.[6] Clark, R.A. Ghosh, K. Tonnesen, M.G. (2007). Tissue engineering for cutaneous wounds. J Invest

Dermatology. 127, 1018-1029.10.1038/sj.jid.5700715[7] Finucane, M. L., & Williams, A. E. (2011). Psychosocial and cultural factors affecting judgments and

decisions about translational stem-cell research. In Translational stem cell research: Issues beyond the debateon the moral status of the human embryo (pp. 391-398). Totowa, NJ: Humana Press.[8] Cao, E., Chen, Y., Cui, Z. and Foster, P. R. (2003), Effect of freezing and thawing rates on denaturation

of proteins in aqueous solutions. Biotechnol. Bioeng., 82: 684690.[9] Pagn R, Mackey B. Relationship between Membrane Damage and Cell Death in Pressure-Treated Es-

cherichia coli Cells: Differences between Exponential- and Stationary-Phase Cells and Variation amongStrains. Applied and Environmental Microbiology. 2000;66(7):2829-2834.[10] Aguado BA1, Mulyasasmita W, Su J, Lampe KJ, Heilshorn SC. Improving viability of stem cells duringsyringe needle flow through the design of hydrogel cell carriers. Tissue Eng Part A. 2012 Apr;18(7-8):806-15.doi: 10.1089/ten.TEA.2011.0391. Epub 2011 Dec 20[11] Walker PA, Jimenez F, Gerber MH, Aroom KR, Shah SK, Harting MT, Gill BS, Savitz SI, Cox CS.,Jr Effect of needle diameter and flow rate on rat and human mesenchymal stromal cell characterization andviability. Tissue Eng Part C Methods. 2010;16:989997.[12] Aguado BA1, Mulyasasmita W, Su J, Lampe KJ, Heilshorn SC. Improving viability of stem cells duringsyringe needle flow through the design of hydrogel cell carriers. Tissue Eng Part A. 2012 Apr;18(7-8):806-15.Epub 2011 Dec 20[13] Chen, X., & Thibeault, S. (2013). Effect of DMSO Concentration, Cell Density and Needle Gauge on theViability of Cryopreserved Cells in Three Dimensional Hyaluronan Hydrogel. Conference Proceedings: An-nual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineeringin Medicine and Biology Society. Conference, 2013, 62286231.[14] Finucane, M. L., & Williams, A. E. (2011). Psychosocial and cultural factors affecting judgments anddecisions about translational stem-cell research. In Translational stem cell research: Issues beyond the debateon the moral status of the human embryo (pp. 391-398). Totowa, NJ: Humana Press.

21

F Proposal

Document begins on the next page.

22

Design Team 8 Project Proposal

A Novel Stem Cell Delivery Device

Michael Clark, Angelica Herrera, Arianne Papa, Michael Mow, and Jack JungProject Sponsor: Dr. Luis Garza∗

Revision: September 30, 2014

An intradermal injection. Source: Novosanis.

Abstract

To facilitate consistent and accurate placement of skin stem cells at the dermoepidermal junction, weplan to design, develop, and test a novel intradermal stem cell delivery device. Although this deliverydevice has a wide range of applications in the field of intradermal cellular therapies, the developmentof the device will be examined within the context of an existing clinical trial taking place at JohnsHopkins Hospital: an investigational stem cell treatment for amputees that aims to initiate growth ofpalmoplantar skin on amputees’ stumps. The device will be designed to deliver stem cells at adjustabledepths and volumes as determined by the physician administering the therapy. The nature of the clinicalneed requires the device to effectively deliver cells at a wide variety of locations. The device will deliverthe cells in such a way as to maximize cell viability and treatment sterility. Patient comfort and devicecost will also be considered and optimized. The device will be tested in vitro in a variety of skin models,and eventually in vivo as part of the aforementioned clinical trial. Valuable subjective feedback will alsobe obtained through the use of several IRB-approved surveys.

∗Assistant Professor, Johns Hopkins Medicine Department of Dermatology, lag@jhmi.edu

Contents

1 Clinical Background 3

2 Clinical Trial 4

3 Standard of Care 4

4 Clinical Problem 5

5 Clinical Need 5

6 Project Goals and Design Constraints 56.1 Project Goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66.2 Design Constraints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

7 Existing Solution Landscape 87.1 Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87.2 Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

References 11

A Microneedle Design Specifications 12

B Current Eight-Week Plan 14

2

1 Clinical Background

Human skin is broken up into three main layers – the epidermis, the dermis, and the subcutaneous tissue(NCI, 2014). Figure 1 depicts the basic anatomy of the skin. The epidermis is the outermost layer, andits thickness varies by location. It is only 0.05mm thick on the eyelids, and is 1.5mm thick on the palmsand the soles of the feet. Keratinocytes are cells found predominantly in the epidermis and primarilyfunction as a barrier against the exterior environment.

Figure 1: Basic anatomy of the skin. Image source:SEER, National Cancer Institute.

In humans, the palmoplantar epidermis is a highly specialized tissue found on the palms and solesthat expresses a wide range of keratins. This palmoplantar (“volar”) skin has different cellular propertiescompared to nonpalmoplantar skin. Figure 2 highlights these differences. This palmoplantar tissue issubjected to the highest degree of mechanical stress that the body is exposed to from extrinsic factors(Fu et al., 2013)

Figure 2: Differences in cellular properties betweenvolar/palmoplantar skin and non-palmoplantar skin.

3

Keratin 9 (KRT9) is a protein that is found only in the palmoplantar epidermis of palms and soles. Ithas been found that KRT9 is required for terminal differentiation as well as maintaining the mechanicalintegrity and structural resilience of the palmoplantar epidermis. Since KRT9 is exclusively expressed inthe palmoplantar epidermis, it can be used as a differentiation marker of palms and soles (Yamaguchi etal., 1999).

Directly below the epidermis is the dermal skin layer, the thickest of the three layers of the skin (1.5to 4.0mm). The dermis makes up approximately ninety percent of the total thickness of the skin. Itconsists of dermal fibroblasts that produce collagen and extracellular matrix components. These cellsalso generate connective tissue and allow the skin to recover from injury by aiding in wound healing.Derived from mesenchymal stem cells within the body, dermal fibroblasts can be further differentiated.

Epithelial-mesenchymal interactions play a key role in the growth and differentiation of keratinocytes.Dermal fibroblasts are directly involved in regulating and controlling epithelial (keratinocyte) function bysecreting diffusible growth factors (Coulomb, Lebreton, & Dubertret, 1989). It has been determined thatKRT9 can be regulated by extrinsic signals from dermal fibroblasts (Yamaguchi et al., 1999). Since thedermal layer determines the phenotypic expression of the epidermal layer, there is great potential to usethese cellular interactions to control the characteristics of the epidermal cells. In order to use epithelial-mesenchymal interactions to differentiate epidermal cells, it is necessary to have a full understanding ofeffective delivery techniques of stem cells to a target skin region for use in stem cell therapy.

2 Clinical Trial

Dr. Luis Garza, a dermatologist at The Johns Hopkins Hospital, has been investigating the use of site-specific autologous fibroblasts to alter skin identity. It has been shown that skin identity can be alteredby obtaining tissue samples from the desired skin type on the patient, culturing the tissue to expand thefibroblasts, and transplanting these cells into the target region on the patient (see Garza, et al. in Section7.2). Garza’s new procedure for injecting fibroblast cells into the skin begins by culturing autologousfibroblast cells with keratinocytes obtained from the epidermis of human foreskin in vitro and freezingthe cells in a solution of DMSO and hetastarch. In order to adhere to FDA regulations and to minimizecontamination, the cells are then injected into the skin in the same solution in which they are frozen(Garza, 2014).

Currently, injections are performed with a needle and syringe. The ideal injection location is thoughtto be at the dermoepidermal junction. Cells must remain sterile, viable, and as concentrated as possiblewithin the tissue. Clinical trials of this procedure will begin in Fall 2014 (Garza, 2014).

Garza’s treatment has the potential to be used for scars, discolored skin, rashes, ulcers, and alopecia.Currently, Garza is working on a clinical trial to inject fibroblast cells into the stump sites of amputeeswho wear prosthetic devices. The study aims to change the phenotype of the stump site skin fromnonvolar to volar. This transformation will prevent skin degradation and provide a better skin-deviceinterface.

3 Standard of Care

In the United States, an estimated 185,000 people undergo an upper or lower limb amputation eachyear. Studies show that about 54% of these amputations arise from dysvascular disease, 45% due totrauma accounts, and less than 2% due to cancer (Ziegler-Graham, MacKenzie, Ephraim, Travison, &Brookmeyer, 2008).

Around 50% of all amputees who wear a lower limb prosthesis report some type of skin degradation attheir stump site due to their prosthetic device. In a study of 247 Vietnam War veterans with amputations,48.2% of patients reported skin breakdown, 39% reported pressure ulcers, 25% reported infection, 25%of patients reported scars and wounds, 21.8% reported rashes, and 21% reported abrasion of the skin

4

(Meulenbelt, Geertzen, Jonkman, & Dijkstra, 2011). These skin conditions are believed to be causedprimarily by friction and mechanical trauma from the prosthesis. Many patients report that these skincomplications lead to substantial pain and discomfort. Examples of these skin complications are depictedin Figure 3. The current standard of care for this problem involves using bandages on the stump site, oraltering or replacing the prosthesis. When those treatments fail, prosthetic abandonment can occur.

Figure 3: Common complications arising from pros-thetic use. Image source: Procellera.

Common interventions include the use of antibiotics for superinfection, emollients and topical corti-costeroids for contact dermatitis, and surgery for epidermal inclusion cysts. Various companies, such asProcellera, provide wound care for amputees, especially athletes who demand the highest performancefrom their prosthetic device. Procellera provides a wound dressing that takes advantage of microcurrenttechnology to prevent the growth of harmful bacteria in the stump dressing and enhance the rate ofwound healing. These treatments only alleviate the problem for the patient. However, Garza’s noveltreatment to inject stem cells into the stump site to create durable, volar skin will prevent the occurrenceof skin degradation (Yamaguchi et al., 1999).

4 Clinical Problem

Currently, physicians have difficulty delivering stem cells to the dermoepidermal junction at consistentdepths. Providers rely on tactile feedback and previous experience to deliver the cell suspension to thedesired location. As a result, variance is inherent in the treatment administration procedure. In order tostandardize the clinical trial data, the stem cell delivery procedure must be as objective and repeatableas possible. A novel stem cell delivery device capable of delivering specific volumes of a cell suspensionat precise, repeatable depths would be very useful.

5 Clinical Need

In patient populations receiving skin stem cell therapies, there is a need for a device that allows physiciansto deliver stem cells to target intradermal regions at adjustable depths and volumes with minimal riskof contamination or damage to the stem cells.

6 Project Goals and Design Constraints

The need specifications for the novel stem cell delivery device are reported below as project goals and de-sign constraints. Project goals reflect desirable project outcomes. Design constraints describe prototypespecifications and testing procedures that will shape the final design.

5

6.1 Project Goals

Goal Reasoning Assessment

Increase Patient Comfort Each year, 11% of Americans whoreceive an amputation abandontheir prosthetic device. Prostheticabandonment is a directconsequence of patient discomfortsecondary to prosthetic use.Increasing patient comfort willreduce prosthetic abandonment.

IRB-approved survey to assesspatient comfort after receiving theskin stem cell therapy. Assessingthis project goal is the primaryresponsibility of Dr. Garza, whooversees the biological andtherapeutic aspect of the project.

Decrease Procedure Pain Cell delivery via hypodermicneedle (the current standard ofcare) can be painful to patients.Reducing the pain of theprocedure will increase patientsatisfaction and lead to morepositive patient outcomes.

IRB-approved survey to assesspatient pain during and after thestem cell delivery takes place.Procedure observation todetermine patients immediatereaction to the delivery procedure.

Increase Cell Viability Stem cells can burst if subjected tohigh shearing forces, such as thoseexperienced when cells are forcedthrough a small needle. Anyconsiderable strain, such as highpressure, placed on the cells willresult in a reduction in cellviability. For this reason,hypodermic needles smaller thantwenty-five gauge are thought tobe damaging to the stem cells.

A physical skin model will bedeveloped to test cell viability. Amock delivery procedure will beperformed on the model, and cell(or cell analogue) growth will bemonitored. Within the context ofthe clinical trial, post-injectionpatient follow-up will allow volarskin growth (and thus cellviability) to be observed.

Increase Treatment Sterility The risk of cells becomingcontaminated increases as they aretransferred from one container toanother. Ultimately, the completeddevice should require a minimumnumber of container transfers.

A physical skin model will bedeveloped, and a mock cell (or cellanalogue) delivery procedure willtake place. Cell growth and skincontamination can then bemonitored using basic wet labtechniques. Within the context ofthe clinical trial, post-deliveryfollow-up with patients will helpdetermine treatment sterility.

Decrease Device Cost The stem cell therapy needs to bean accessible, viable option forpatients. Reducing the cost toproviders and heath care systemswill encourage them to utilize thetechnology.

The cost of the novel deliverydevice will be compared to thecost of the current standard ofcare. The device cost will also becompared to the cost of the stemcell therapy, which is estimated tobe between $1000 and $5000 USD.Health care providers andadministrations will be surveyed todetermine if costs are prohibitive,competitive, and/or incentivising.

6

6.2 Design Constraints

Constraint Reasoning Assessment

A Repeatable, ConsistentDelivery Procedure

The specifics of the stem celldelivery procedure is currentlyunder the discretion of the doctorperforming the injection. Changesin the procedure may arise fromvariation from patient to patientvariation as well as variation fromdoctor to doctor. The deliverydevice must perform consistentlyand repeatably despite thesevariations.

An IRB-approved survey will beemployed to obtain feedback fromphysicians. A physical skin modelwill be sourced and/or developed,which will allow physicians to trythe device and offer feedback. Themodel will also be used to verifythe correct placement of the stemcells. Post-delivery follow-up withpatients treated with the noveldelivery technology will becompleted in order to comparetheir outcomes with “currentstandard of care” patientoutcomes.

Adjustable Depths Different locations on the bodyhave different epidural thicknesses.Epidural thickness may also varyfrom patient to patient. Thedevice will need to adjust to thesevarying depths (50-150micrometers), with a resolution ofat least 1 micrometer.

An IRB-approved survey will beused to assess physicians’ sense ofdepth control. Physical skinmodels of different epiduralthicknesses will be employed totest the accuracy and precision ofthe device. Post-delivery follow-upwith patients will be conducted toevaluate stem cell placement.

Adjustable Volumes The novel device must be able tocompete with the existing syringemethod used in clinical trials,which has the ability to deliverspecific volumes of the stem celltreatment. The final device willneed to be capable of delivering37 × 106 cells in 750µL ofcryopreservation fluid.

An IRB-approved survey will beused to assess physicians’ sense ofvolume control. Physical skinmodels will be employed to assessthe volume of cell suspensiondelivered to the epidermis-dermisinterface.

Reusability Device cost is an importantconsideration, and can be reducedif all or part of the device isreusable.

An IRB-approved survey can beconducted to assess physicians’thoughts on reusable components.Cost analyses and a continuingsurvey of existing technologies willguide decisions relating to devicereusability.

Delivery Locations The final device must be able totarget different stump locations onthe body of multiple sizes,contours, and underlyingstructures. If the device is to beused outside Garza’s clinical trial,the device will need to work inlocations other than stump skin.

Various skin models will be usedto evaluate the efficacy of thedevice when confronted with avariety of epidural thicknesses,surface contours, and underlyingstructures.

7

7 Existing Solution Landscape

7.1 Technologies

Stem cell therapy is an emerging field in medicine. Despite its rapid growth, there has been little devel-opment in the area of intradermal stem cell delivery technologies in recent years. Instead, doctors mustdepend on traditional drug delivery methods to administer cellular therapies. Fortunately, traditionaldevices for general transdermal and intradermal drug delivery are plentiful. Research has been con-ducted to determine how these existing drug delivery technologies can be modified and adapted to suitthe emerging challenge of intradermal stem cell delivery. Examples of these existing technologies includehypodermic needles, microneedle arrays, and liquid jet injectors. A summary of these technologies isdisplayed in Figure 4.

Figure 4: A visual survey of different injection technolo-gies.

Macromolecular drug delivery across the skin is primarily accomplished using a hypodermic needleand the Mantoux technique. Not only is this the cheapest device on the market but also the most widelyavailable around the world. Hypodermic needles come in various sizes ranging from 6 (I.D. = 4.39 mm)to 34 gauge (I.D. = 0.08 mm) . The smallest gauge that can be feasibly implemented is 25 (I.D. = 0.26mm) as any larger gauge would increase the shear stress on the cells during extensional flow. Hypo-dermic needles possess major drawbacks, such as acute cell death due to shear stress, inconsistencies indelivered dosage, and safety concerns (such as accidental needle sticks) (Kis, Winter, & Myschik, 2012).Providers typically utilize standard luer-lock syringes. Garza has developed a novel syringe that allowsthe stem cell suspension to be injected directly from a cryogenic storage tube. This device has not beenmanufactured or used to deliver any clinical therapies.

Microneedle arrays are micron-scale needles used for transdermal drug delivery. Typically, micronee-dles are fabricated as an array of up to hundreds of microneedles over a base substrate. There are fourmain classifications and designs, listed here in the order they appear in Figure 5: solid microneedles thatpierce the skin to make it more permeable, solid microneedles coated with dry powder drugs or vaccinesfor dissolution in the skin, microneedles prepared from polymer with encapsulated vaccine for rapid orcontrolled release in the skin, and hollow microneedles for injections (Arora, Prausnitz, & Mitragotri,2008). Due to the relatively large size of the stem cells, microneedles will be difficult to adapt for stemcell injection. Though clinical trials are ongoing, there has so far been no significant adverse reactionsto microneedles other than minor pain and mild skin irritation, which occur in most manual injectionmethods. A complete list of materials used and design specifications is attached in the appendix.

8

Figure 5: A visual summary of microneedle technologies.Image source: Arora et al., 2008.

Liquid jet injections employ a high-speed jet of liquid that punctures the skin and delivers drugswithout the use of a needle. The basic design of commercial liquid jet injectors consists of a powersource, piston, drug-loaded compartment and a nozzle with an orifice size typically ranging between150 and 300 micrometers. Drug delivery via jet injection takes place in two phases. Upon triggeringthe actuation mechanism, the power source, either a spring or compressed gas, pushes the piston whichimpacts the drug-loaded compartment, leading to a quick increase in pressure. This forces the drugsolution through the nozzle as a liquid jet with a velocity ranging between 100 to 200 meters per second(Baxter & Mitragotri, 2005). The jet punctures through the skin and initiates hole formation as shownin Figure 6. The second phase then begins with a multi-directional jet dispersion from the end point ofpenetration. However, concerns regarding cell viability and the forces involved with the expulsion of thedrug solution at high pressures make this approach infeasible in its current state (Aguado, Mulyasasmita,Su, Lampe, & Heilshorn, 2012).

Figure 6: A visual summary of liquid jet injector tech-nology. Image source: Arora et al., 2008.

9

7.2 Patents

A summary of relevant patents and devices is presented below. Due to length concerns, the full text ofthe patents are omitted from this document.

Arora, et al. — Allergan, Inc.Soft tissue augmentation by needle-free injectionGranted Patent, US 8021323 B2The invention relates to needle-free apparatus that can be used to augment soft tissue. More specifically,the needle-free injectors of the present invention allow injection of more viscous materials such as col-lagen, hyaluronic acid, and other polymers that are useful as dermal fillers. The needle-free injectors ofthe present invention allow injection of such materials to fill the undesired lines, wrinkles, and folds of apatient. The present invention also relates to kits comprising such needle-free injectors and a quantity ofdermal filling material. In addition, the present invention relates to methods of augmenting soft tissueusing needle-free apparatus.

Mudd, et al. — Allergan IncModular Injection DeviceGranted Patent, US 8480630 B2, EP 2571550 B1A modular injection device for administration of dermal filler compositions is provided. The injectiondevice may include a handheld injector unit including a drive unit, the drive unit configured to applyan extrusion force to a fluid; a control unit remote from the injector unit, the control unit configured tocontrol the drive unit; and a cable configured to connect the control unit to the injector unit.

Sheldon et al, — Antares Pharma Inc.Single Use Disposable Jet InjectorGranted Patent, EP 1265663 B1The present invention is directed to a device for delivery of medicament, and in particular to a single usedisposable jet injector.

Heneveld, et al. — Aesthetic Sciences Corp. Apparatus And Methods For Injecting High Viscos-ity Dermal FillersPatent Applications, US 2009/0124996 A, AU 2008/283868 A1, WO 2009/021020 A1A method includes inserting a distal end portion of a needle of a medical injector into a skin of a body. Anenergy source operatively coupled to the medical injector is actuated such that a dermal filler is conveyedfrom the medical injector into the skin through the distal end portion of the needle. The distal end portionof the needle is moved within the skin during the actuating.

Garza, et al. — The Johns Hopkins UniversityMethods For Using Autologous Fibroblasts To Alter Skin IdentityPatent Applications, WO 2013/166045 AlThe present invention relates to the field of autologous fibroblasts. More specifically, the present inven-tion provides methods and compositions comprising autologous fibroblasts and uses thereof to alter skinidentity. In certain embodiments, volar fibroblasts can be expanded for the ability to induce volar skinat the stump site in amputees. In other embodiments, fibroblasts from haired scalp can be expanded toameliorate alopecias.

Replicel Life Sciences, Inc.RCl-02: Dermatology injector deviceNo Patent Found

10

References

Aguado, B., Mulyasasmita, W., Su, J., Lampe, K., & Heilshorn, S. (2012). Improving viabilityof stem cells during syringe needle flow through the design of hydrogel cell carriers. TissueEnginering. Part A., 18 (7–8), 806-815.

Arora, A., Prausnitz, M. R., & Mitragotri, S. (2008). Micro-scale devices for transdermal drugdelivery. International Journal of Pharmaceutics, 364 (2), 227–236.

Baxter, J., & Mitragotri, S. (2005). Jet-induced skin puncture and its impact on needle-freejet injections: experimental studies and a predictive model. Journal of Controlled Release,106 (3), 361–373.

Coulomb, B., Lebreton, C., & Dubertret, L. (1989). Influence of human dermal fibroblasts onepidermalization. Journal of Investigative Dermatology , 92 (1), 122–125.

Fu, D. J., Thomson, C., Lunny, D. P., Dopping-Hepenstal, P. J., McGrath, J. A., Smith, F. J.,. . . Pedrioli, D. M. L. (2013). Keratin 9 is required for the structural integrity and terminaldifferentiation of the palmoplantar epidermis. Journal of Investigative Dermatology .

Garza, L. (2014). Design team 8 interivew..Kis, E. E., Winter, G., & Myschik, J. (2012). Devices for intradermal vaccination. Vaccine,

30 (3), 523–538.Meulenbelt, H. E., Geertzen, J. H., Jonkman, M. F., & Dijkstra, P. U. (2011). Skin problems of

the stump in lower limb amputees: 1. a clinical study. Acta dermato-venereologica, 91 (2),173–177.

NCI. (2014). Seer training modules: Layers of the skin. Online.Yamaguchi, Y., Itami, S., Tarutani, M., Hosokawa, K., Miura, H., & Yoshikawa, K. (1999). Reg-

ulation of keratin 9 in nonpalmoplantar keratinocytes by palmoplantar fibroblasts throughepithelial–mesenchymal interactions. Journal of Investigative Dermatology , 112 (4), 483–488.

Ziegler-Graham, K., MacKenzie, E. J., Ephraim, P. L., Travison, T. G., & Brookmeyer, R.(2008). Estimating the prevalence of limb loss in the united states: 2005 to 2050. Archivesof physical medicine and rehabilitation, 89 (3), 422–429.

11

A Microneedle Design Specifications

12

13

B Current Eight-Week Plan

(Document begins on next page)

14

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