Co-cultivation of cells in a micropatterned configuration

Details Co-cultivation of cells in a micropatterned configuration Co-cultivation of cells in a micropatterned configuration

2000-01-13

2001-04-24

Tate, Christopher R. (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of co-culturing cells

C435S395000, C435S402000, C435S289100, C435S305100, C435S309100, C435S177000

Reexamination Certificate

active

06221663

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to methods for co-cultivating cells in micropatterned formations (e.g., for the production of bioartificial organs).
Co-cultures of hepatocytes with another cell type have been recognized to prolong cell survival rates, maintain phenotype, and induce albumin secretion in hepatocytes. Such co-cultures have been limited by the inability to manipulate or control the interaction of the two cell types in the culture. Generally, to prepare conventional co-cultures, cells of one type are seeded onto a substrate and allowed to attach; cells of a second type then are seeded on top of the cells of the first type. In such co-cultures, parameters such as cell number are controllable, but the spatial orientation of cells within the co-culture is not controlled (Clement, B., et al. “Long-Term Co-Culture of Adult Human Hepatocytes with Rat Liver Epithelial Cells: Modulation of Albumin Secretion and Accumulation of Extracellular Material” Hepatology 4(3): 373-380 (1984); Schrode, W., et al. “Induction of Glutamine Synthetase in Periportal Hepatocytes by Cocultivation with a Liver Epithelial Cell Line” Euro. J. Cell Biol. 53: 35-41 (1990); Michalopoulos, G., et al., In Vitro 15(10): 796-806 (1979); Guguen-Guillouzo, C., et al. “Maintenance and Reversibility of Active Albumin Secretion by Adult Rat Hepatocytes Co-Cultured with Another Liver Epithelial Cell Type” Experimental Cell Research 143: 47-54 (1983); Begue, J. et al. “Prolonged Maintenance of Active Cytochrome P-450 in Adult Rat Hepatocytes Co-Cultured with Another Liver Cell Type” Hepatology 4(5): 839-842 (1984); Agius, L. “Metabolic Interactions of Parenchymal Hepatocytes and Dividing Epithelial Cells in Co-culture” Biochem. J. 252: 23-28 (1988); and Reid, L. et al. “Culturing Hepatocytes and Other Differentiated Cells” Hepatology 4(3): 548-559 (1984)).
SUMMARY OF THE INVENTION
The invention provides methods for producing co-cultures of cells in which at least two types of cells are configured in a micropattern on a substrate. By using micropatterning techniques to modulate the extent of heterotypic cell-cell contacts, it is now possible to modulate (e.g., upregulate or downregulate) metabolic and/or synthetic functions of cells.
Accordingly, the invention provides a method for producing a micropatterned co-culture containing at least two cell types; the method entails:
i) providing a protein-coated substrate, wherein a protein coating the substrate defines a micropattern on the substrate;
ii) contacting the protein-coated substrate with cells of a first cell type suspended in a first cell medium under conditions such that cells of the first cell type bind the protein of the protein-coated substrate, thereby producing a micropatterned cell-coated substrate; and
iii) contacting the micropatterned cell-coated substrate with cells of a second cell type suspended in a second cell medium under conditions such that cells of the second cell type bind the substrate, thereby producing the micropatterned co-culture, wherein one of the cell media is a selective medium and one of the cell media is an attachment medium.
Typically, in practicing the invention, the cells of the first and second cell types are mammalian cells, although the cells may be from two different species (e.g., pigs, humans, rats, mice, etc). The cells can be primary cells, or they may be derived from an established cell line. In an alternative method, one of the cell types is mammalian, and a second cell type is microbial in origin, e.g., fungi or bacteria such as Streptococcus ssp.,
Staphylococcus aureus,
or
Staphylococcus epidermis.
Examples of suitable combinations of cells for producing the co-culture include, without limitation:
a) hepatocytes (e.g., primary hepatocytes) and fibroblasts (e.g., normal or transformed fibroblasts, such as NIH 3T3-J2 cells);
b) hepatocytes and at least one other cell type, particularly liver cells, such as Kupffer cells, Ito cells, endothelial cells, and biliary ductal cells;
c) endothelial cells and smooth muscle cells;
d) tumorigenic parenchymal cells and mesenchymal cells;
e) hematopoietic cells and bone marrow cells (e.g., adipocytes, fibroblasts); and
f) skin cells (e.g., keratinocytes) and fibroblasts. Other combinations of cells also are within the invention.
The substrate on which the cells are grown can be any biologically compatible material to which cells can adhere, such as glass, polymers (such as fluoropolymers, fluorinated ethylene propylene, polyvinylidene, polydimethylsiloxane, polystyrene, polycarbonate, and polyvinyl chloride), and silicon substrates (such as fused silica, polysilicon, or single silicon crystals).
To produce a micropattern of the co-cultured cell types, protein (i.e., a peptide of at least two amino acids) is first adhered to the substrate in order to define (i.e., produce) a micropattern. The micropattern produced by the protein serves as a “template” for formation of the cellular micropattern. Typically, a single protein will be adhered to the substrate, although two or more proteins may be used to define the micropattern (for example, one micropatterned protein may be used to attract one cell type, while a second micropatterned protein is used to attract a second cell type). In practicing the invention, a variety of techniques can be used to foster selective cell adhesion of two or more cell types to the substrate. Included, without limitation, are methods such as localized protein adsorption, organosilane surface modification, alkane thiol self-assembled monolayer surface modification, wet and dry etching techniques for creating three-dimensional substrates, radiofrequency modification, and ion-implantation (Lom et al., 1993, J. Neurosci. Methods 50:385-397; Brittland et al., 1992, Biotechnology Progress 8:155-160; Singhvi et al., 1994, Science 264:696-698; Singhvi et al., 1994, Biotechnology and Bioengineering 43:764-771; Ranieri et al., 1994, Intl. J. Devel. Neurosci. 12(8):725-735; Bellamkonda et al., 1994, Biotechnology and Bioengineering 43:543-554; and Valentini et al., 1993, J. Biomaterials Science Polymer Edition 5(1/2):13-36).
Proteins that are suitable for producing a micropattern are those proteins to which one of the cell types of the co-culture specifically binds under the cell culture conditions used to cultivate the co-culture (i.e., conventional cell culture conditions). For example, hepatocytes are known to bind to collagen. Therefore, collagen is well-suited to facilitate binding of hepatocytes in a micropattern. Other suitable proteins include fibronectin, gelatin, collagen type IV, laminin, entactin, and other basement proteins, including glycosaminoglycans such as heparan sulfate. Combinations of such proteins also can be used.
Typically, in practicing the invention, the cells of the first cell type (e.g., hepatocytes) initially are suspended in an “selective” cell culture medium (e.g., serum-free medium and media that lack “attachment factors”), while the cells of the second cell type are suspended in an “attachment” medium [e.g., a cell culture medium that contains serum (typically 1-10% (e.g., 5-10%)), or one or more “attachment factors” (typically at least 1 ng/ml (e.g., 5-100 ng/ml)) such as fibronectins and other extracellular matrix, selecting, RGD peptides, ICAMs, E-cadherins, and antibodies that specifically bind a cell surface protein (for example, an integrin, ICAM, selectin, or E-cadherin)].
In another method of practicing of the invention, the cells of the second type have intrinsic attachment capabilities, thus eliminating a need for the addition of serum or exogenous attachment factors. Some cell types will attach to electrically charged cell culture substrates and will adhere to the substrate via cell surface proteins and by secretion of extracellular matrix molecules. Fibroblasts are an example of one cell type that will attach to cell culture substrates under these conditions. Thus, the invention also includes a method for producing a micropatterned co-culture containing at least two cell types where

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