BTE 4223DR. MUNIRA SHAHBUDDIN

KULLIYYAH OF ENGINEERING

IIUMLECTURE I

ENGINEERING ASPECT OF ANIMAL CELL AND

TISSUE CULTURE

INTRODUCTION• WHAT IS ACTUALLY ANIMAL CELL CULTURE?The main subject of animal cell technology is to deal with control and modulation of cellular function.Conventional cell biology regards cells and tissues as constituent of animal body.The analyses of differentiated cellular function give the basic information needed to understand composite functions of organs and animals which consist of organized assemblies of various kinds of cells

• Animal cells technology recognizes an animal cells as an independent living organism

EXAMPLES OF TRANSFORMATION FROM CELLS INTO A SYSTEM

• Animal cell technology is very interested in focusing on the construction and functional regulation of engineered cells which are reconstituted so as to express their specific biological function in vitro.

• Animal cell culture technology is a new scientific discipline closely associated with the further development of biotechnological industrial field.

• The research on bio-functional regulation of engineered cells will lead to a much more work and clear understanding of cascades of an in vivo physiological events.

• Biological function of engineered cells can be controlled and modulated by various means which are categorized into three: genetic, physiological and physical means.

CHOOSING THE APPROPRIATE ANIMAL CELL CULTURE MODEL• Why opt for animal cell culture? What is so wrong

with animal sacrifices? • Cost analysis and benefit.• The use of animal cells will reduce the cost for

animal care and housing, less time consuming and much cleaner.

• Effective and easy.• Rules and regulation of economic block and

countries

• The test for cosmetics and household products should not be so cruel but rather simple, easy, rapid and effective.

Left: Inflammatory test on rabbit’s eye and Right: on human tissue engineered skin

CONSIDERATIONS FOR ANIMAL CELL CULTURE• The vision or model of an animal cell culture system as difficult,

inefficient and uneconomic is a result of the perception that it requires expensive media, easily contaminated and difficulties for scaling-up process.

• The implementation of Good Manufacturing Practices has led to the almost complete elimination of contamination.

• There are considerations that should be taken while doing cell culture: oxygen supply and demand, oxygen delivery system, air flow rates, bubble diameter, oxygen transfer rate, bubble residences, cellular physiologies, oxygen solubility, pH, and etcetera.

WHY CONSIDER?

1. Think of a system that mimic human physiology, what do you require?

2. What will be the effect of the system microenvironment to cellular physiology and biological effects?

3. What is cellular microenvironment?

Designing an experiment or model encourage us to work outside the conventional practices. In general it provides us with the basis for further investigations to improve our understanding on cellular biology and system.

REQUIREMENTS FOR ANIMAL CELL CULTURE• Cell culture media• Incubator• Laminar hood• Laboratory apparatus – pipettes, petri dishes, beaker etc.• Disinfectant – bleach and 70% ethanol• Cryopreservation medium

An easy way to obtain epidermal skin…….

• When cells from adult foreskin epidermis were cultured in vitro for 7–10 days, small fusiform cells appeared (P0 7d), while other cell types almost completely disappeared. With prolonged incubation, these small fusiform cells continued to increased in number (P0 12d and P0 15d). (B): Before passage 10, most cells were short and spindle-shaped (P2 2d). Within the first 30 passages, cells generally formed a single layer within 1.5–3 days after replating (P2 3d). (C–E): After passage 10, most cells had short spindle or regular spindle morphologies, with two to three processes projecting from the soma (C, round figure on the left, see arrow), while a minority had multiple processes (C, round figure on the right, see arrow). In some specimens, most of the hEMSCPCs had short processes, while only a minority had long and slim processes (D, round figure on the left, see arrow). In other specimens, however, most of the hEMSCPCs had long and slim processes. (F): When hEMSCPCs formed a monolayer, they took on a swirling arrangement resembling that of mesenchymal stem cells.

Reference: http://www.nature.com/articles/srep01933

Reference to the paper: Organotypic liver culture models: Meeting current challenges in toxicity testing. LeCluyse EL, Witek RP, Andersen ME, Powers MJ - (2012)http://openi.nlm.nih.gov/detailedresult.php?img=3423873_btxc42-501-f8&query=null&req=4&npos=-1

fig5: Major nonparenchymal cell types of the liver. Top row: CD-31 staining of liver sinusoidal endothelial cells (LSEC) lining vascular walls of whole liver (A), scanning electron micrograph of the endothelial lining of the liver sinusoids showing extensive patches of fenestrae (arrows) (B), and primary LSEC showing typical morphology in vitro (C). Middle row: HSC (GFAP) in normal liver (D), myoflbroblastic HSC ((iSMA) in flbrotic liver (E), and isolated qHSC showing storage of vitamin A as bright “floating” vesicles within the cell body. Upon activation or injury the HSC undergo extensive morphological and biochemical changes, which include the synthesis, secretion and restructuring of ECM molecules. Bottom row: Kupffer cells (KC) showing their dynamic morphology (G), their identification with CD68 showing extended projections on the cell bodies used for contact with other cells (H), and a magnified view showing KC loaded with vesicles containing cytokines and other secretory factors (I).

fig8: Hepatic cell culture model systems represented in “Advanced organotypic culture technologies” (A) Perfusion array liver system (PEARL) (Lee et al., 2007), (B) bioengineered micropatterned liver platform (Khetani and Bhatia, 2008), (C) biochip dynamic flow system (Chao et al., 2009; Novik et al., 2010; Maguire et al., 2009), (D) 3-D liver tissue culture scaffold (Sibanda et al., 1993; Sibanda et al., 1994; Naughton et al., 1994), and (E) 3-D scaffolds with dynamic flow (Sivaraman et al., 2005; Domansky et al., 2010).

• For the next class, I would like you to read on the differences between animal and animal cell experimentation for discussion.

• Everyone is required to read on animal cells and tissue laboratory manuals and regulations.