Novel Method for Precise Co-Culture of Fibroblast and Osteosarcoma Cells to Investigate Tumor Development

1Schiele, N R; 1Carr, B M; 1Chrisey, D B; +1Corr, D T1Rensselaer Polytechnic Institute, Troy, NY

corrd@rpi.edu

Introduction: Tumor development and growth is dependent on cellsignaling and communication, which cannot normally be controlledduring in vitro investigation, especially with homogenously mixed co-cultures. Affecting the modes of cellular signaling (direct cell contact,paracrine, and endocrine), by regulating nearest neighbor cell type andgeometric cellular proximity is essential to gain a more fundamental andcomplete understanding of cancer metastasis and tumor development, aswell as normal extracellular interactions. In vivo, a tumor typicallypresents as a mass of cancerous cells surrounded by healthy tissue, butthe importance of this histologic structure is rarely considered whenstudying cells in vitro. Since the cellular composition and arrangementof an in vitro culture will affect the mode of communication and thetypes of cells in communication, it is critical to control the location ofcancer cells (and, thus, the cell signaling) in relation to other normal andnon-cancerous cells. Such control would provide the ability to replicatedesired tumor geometry in in vitro culture, at least in two dimensions,and the cellular response and intracellular cues could be monitored andinvestigated as a function of structural arrangement and geometriclocation. In order to study the effect of cell proximity and neighboringcell type on the cellular decisions and response, we have developed atool for precisely patterning multiple cell types in co-culture. This laser-based cellular patterning technique can create cultures with precise two-dimensional spatial arrangement of multiple cell types and allowsnormal growth following transfer to enable further investigation ofcellular response and development in co-cultures.

Methods: A laser-based method was developed to pattern multiple celltypes in specific arrangements to create co-cultures in which the cellspacing and neighboring cell type are precisely controlled. Using amatrix assisted pulsed laser evaporation direct write (MAPLE DW)approach adapted for multiple cell types, allowed for patterned co-cultures to be created to investigate the effect of cell proximity andneighboring cell type on cellular interactions. To create these co-cultures, both fibroblast cells and MG-63 osteosarcoma cells (grown instandard cell culture conditions using cell culture media composed of89.5% DMEM, 10% FBS, and 0.5% penicillin/streptomycin) werepartially encapsulated in gelatin on the bottom side of two separatequartz ribbons. An ArF pulsed laser (Teosys, Crofton MD) operating at193nm coupled with CAD/CAM capabilities and a motorized stage(Figure 1) was then used to transfer cells from each quartz ribbon to apoly-l-lysine and gelatin coated receiving Petri dish. The laser systemallows for patterns to be developed in a commercial CAD package or g-code, and converted into machine code to direct precise cellularpatterning. Customized patterns can be rapidly developed for variousstudies and cell types (Figure 2). Switching between multiple quartzribbons provides for the patterning of multiple cell types, and theintegrated optical systems allows for visual verification of cell transferas well as specific cell targeting. The number of cells in each transferredspot can be varied, from a single cell to over 300 cells, throughadjustment of the laser beam diameter.

Figure 1: Schematic of MAPLE DW technique used to precisely patterncells with spatial control, including fibroblast and MG-63 co-cultures.

Figure 2: Example of alternating pattern (left panel) and separatedpattern (right panel) used to explore the interactions between fibroblastcells (blue) and MG-63 osteosarcoma (red). Co-cultures produced fromthese input patterns are exhibited in Figure 3.

Figure 3: (Upper Panel) Co-culture pattern of alternating MG-63osteosarcoma and fibroblast cells, 30 minutes after transfer. From left toright the pattern is: MG-63, Fibroblast, MG-63, Fibroblast.(Lower Panel) Co-culture pattern of separated MG-63 and fibroblast, 3hours after transfer. From left to right the pattern is: MG-63, MG-63,Fibroblast, Fibroblast.

Results: We have demonstrated the ability of this method to patternfibroblast and MG-63 cells with great precision, in specific locationswithin a co-culture (Figure 3) based on the predetermined arrangements(Figure 2). This cell writing technique produces viable cells in precisepatterns, which allows for co-cultures to be created that replicate adesired tumor histology, thereby recapitulating the interactions of thetumor’s constituent cells. Furthermore, this technique can be easilyadapted and expanded for further study of cellular interactions.

Discussion: These initial results demonstrate the ability of this cellulardirect write technique to precisely pattern multiple cell types on thesame surface, thereby enabling normal cellular interaction andsubsequent investigation of the cellular effects. Cellular communicationcan be regulated by altering cell neighbor and geometric spacing, whichpresents new investigative opportunities. Using multiple cell types inspecific patterns may better reproduce a cellular environment in vitrothat mimics in vivo conditions that are essential to understandingmechanisms of cancer survival and growth. We are currently using gelelectrophoresis and quantitative polymerase chain reaction, q-PCR, toanalyze the effects of geometric spacing and neighboring cell type inosteosarcoma-fibroblast co-cultures. If it is shown that the cell behaviorand function are significantly affected by their compositionalarrangement in these cultures, a fundamental change in the way cellculture is used to study tumor behavior could follow.

Poster No. 1641 • 56th Annual Meeting of the Orthopaedic Research Society