Types of aggregation cultures
Natural aggregates
While various types of scaffolds are essential to shape the engineered tissue’s morphology and physiological function, it has been realized that scaffolds are not necessarily premade as cells can secrete their ECM and can generate their own scaffolds. This observation and the ability of cells to adhere to each-other and various surfaces (Figure XI-1) are exploited in aggregation cultures.
Figure XI-1: Cell adhesion Cell-cell
interactions
Cell-matrixinteractions
Soluble ECM
Integrins
Static ECM Cadherins
76 The project is funded by the European Union and co-financed by the European Social Fund.
Suspension aggregates. Similarly to several other tissue types, monolayer cultures of chondrocytes dedifferentiate while cell-matrix interactions that control multiple differentiation processes can preserve chondrocyte differentiation. In suspension culture, phenotypically stable chondrocytes disperse in the culture medium first, then proceed to form aggregates. This aggregation process is thought to reproduce in situ interactions, since phenotype that bears high similarity to the native cartilage environment is maintained in aggregate or pellet cultures. Studies of the specific molecular mechanisms involved in forming chondrocyte aggregates elucidated both cell-cell and cell-matrix interactions during the process. The molecular mechanisms have been shown not to involve (1) direct cell-cell interactions rather (2) aggregating to a collagen II and cartilage oligomeric protein (COMP) rich matrix as well as (3) to β1-integrin, which binds a triple helical GFOGER sequence found in collagens. β1 integrin has also known binding properties to collagen type II, namely integrins α1, α2, α10, or α11 β1. At later stages cell-matrix and cell-cell interactions in cartilage are of crucial
importance in mediating cartilage development, homeostasis, degradation and cell survival. These interactions have been extensively studied in the context of limb bud mesenchymal cell condensation, an early stage of cartilage development that is believed to be mediated by fibronectin interactions. After the initial condensation stage, cell-cell contacts are strengthened by direct cell-cell-cell-cell adhesion via the homotypic cell-cell adhesion molecules, NCAM and N-cadherin. Perturbation of NCAM and N-cadherin activity results in reduced or altered cell aggregation and chondrogenesis in vitro and in vivo. However, NCAMs and N-cadherins then disappear from the forming cartilage and are not expressed by differentiated chondrocytes in situ. In healthy adult cartilage, there are no cell-cell contacts, but rather functional cell-matrix contacts, primarily
integrin-Aggregation cultures
Identification number:
TÁMOP-4.1.2-08/1/A-2009-0011
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Understandably, administration of collagen type I to chondrocytes in suspension increases aggregation, whereas collagenase inhibits it.
Gravity aggregates – Hanging drop (HD) cultures. For in vitro aggregation of various cell types hanging-drop culture can be used (Figure XI-2 and Figure XI-3).
Figure XI-2: Microgravity culture (hanging-drop) I
Cavity slide
Sample placed on coverslip with loop
Oil drop Vaseline
180°
78 The project is funded by the European Union and co-financed by the European Social Fund.
Figure XI-3: Microgravity culture (hanging-drop) II
Microgravity culture was developed where cells are inoculated into a drop of fluid attached to a coverglass inverted over a hollow slide. To aid this kind of cellular aggregation, sterile, specific plastic plates (Terasaki plate) have been designed. 60-well and 72-well Terasaki plates are made of medical-grade polystyrene. The volume of each well is approximately 10 μl, therefore adding 20 μl to each well then turning them upside down creates a hanging drop that is kept in place by the capillary effect. Within the drop of fluid, cells of the suspension can gently sediment and form an aggregate. As the volume of the well limits the amount of nutrients in the culture, once the aggregate is formed, the microtissue needs to be transferred to a larger container.
Tissue engineering application of both suspension and hanging drop aggregation cultures has been successful in the generation of embryoid bodies (EB). From enzymatically digested clumps of undifferentiated human embryonic stem cell (hESCs) colonies, embryoid bodies (EBs) are formed following in vitro aggregation.
180° 180°
180°
Time (days)
Outgrowth of plated EBs and spontaneous differentiation into cell types of all three germ layers
0
2
5
Aggregation cultures
Identification number:
TÁMOP-4.1.2-08/1/A-2009-0011
79 Aggregation of hESCs allows for the spontaneous differentiation of cells representing endoderm, mesoderm, and ectoderm lineages. Both aggregation methods allow addition of exogenous growth factors or overexpression of regulatory genes to enhance the generation of differentiated cells. Added factors include: hematopoietic cytokines in combination with the mesoderm inducer BMP-4 that can generate up to 90% of CD45+ hematopoietic cells with colony-forming unit (CFU) activity.
Gravity aggregates – Spun-down aggregates. To decrease proximity of cells and increase the potential for forming aggregates, cell suspension cultures of purified rodent or human cells are spun together to create aggregated organ cultures. These types of aggregated organ cultures are easily assembled, cellular composition can be carefully selected, cell types can be manipulated (gene expression modification, physiological dies, etc) creating a well controlled environment for research studies and to model disease processes.
Synthetic cell aggregation
In synthetic cell aggregation the main concept is to create a polymer bridge between two cells that holds them together (Figure XI-4).
Figure XI-4: Synthetic cell aggregation
Aggregated cells Cells
Bifunctional polymer
80 The project is funded by the European Union and co-financed by the European Social Fund.
The polymer can also be substituted with cell adhesive molecules that following cross-linking, aid agglomeration of cells. Various materials are used for synthetic cell aggregation including chitosan, modified polyethylene glycol (PEG), lectins and derivatives, PLGA nanospheres, etc. Chemical cross linking of cells is also used by applying biotinylated cell cross liners (Figure XI-5).
Figure XI-5: Biotinylated cell cross-linking
Cells can also be seeded on high cellular density onto porous scaffolds. Confined to the pores, cells are forced to aggregate. Placed into bioreactors movement of the media facilitates delivery of nutrients and oxygen that can enhance proliferation of cells leading to further aggregation.
Avidin
Multicellular aggregate Biotin
hydrazide
Periodate tested cells
Identification number:
TÁMOP-4.1.2-08/1/A-2009-0011
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