Histotypic culture

Animal biotechnology

1. Histotypic Culture BHAVYASHREE P V II M.SC BIOTECHNOLOGY ALVA’S COLLEGE MOODBIDRI

2. Three main types of techniques Organ culture- In this type of culture, the whole organs or small fragments of the organs with their spatial and intrinsic properties are used in culture. Histotypic culture- The propagated cell lines grown in three dimensional matrix to high density represent histotypic cultures . Organotypic cultures- C ells of different lineages are combined in experimentally determined ratios and spatial relationships to recreate a component of the organ under study.

3. Histotypic culture: Growth and propagation of cell lines in three-dimensional matrix to high cell density It is possible to use dispersed monolayers to regenerate tissue like structures

4. Commonly used techniques are: Gel and sponge techniques Hollow fibers Spheroids Rotating chamber systems Immobilization of living cells in alginate Filter well inserts Cultures of neuronal aggregates

5. Gel and sponge techniques Leighton first demonstrated that both normal and malignant cells penetrate cellulose sponge The gel or sponge are used which provide the matrix for the morphogenesis and cell growth. The cell penetrates these gels and sponges while growing. Collagen gel provides a matrix for the morphogenesis of primitive epithelial structures . Many different types of cell can be shown to penetrate such matrices and establish a tissuelike histology The kidney epithelial cell line MDCK responds to paracrine stimulation from fibroblasts by producing tubular structures, but only in collagen gel

6. Matrigel . Matrigel is a commercial product derived from the extracellular matrix of the Engelbreth –Holm–Swarm (EHS) mouse sarcoma T hat has been used for coating plastic but can also be used in gel form . It is composed of laminin , collagen, fibronectin , and proteoglycans with a number of bound growth factors, although it can be obtained in a growth factor depleted form U sed as a substrate for epithelial morphogenesis formation of capillaries from endothelial cells and in the study of malignant invasion. It’s a complex and not completely defined matrix and can also inhibit some morphogenetic events, such as hepatocyte growth factor (HGF)-induced tubulogenesis of MDCK cells

7. Hollow fiber techniques Hollow fibers are used which helps in more efficient nutrient and gas exchange In recent years, perfusion chambers with a bed of plastic capillary fibers have been developed to be used for histotypic type of cultures The cells get attached to capillary fibers and increase in cell density to form tissue like structures

8. Contd … The fibers are gas- and nutrient permeable and support cell growth on their outer surfaces. Medium , saturated with 5% CO 2 in air, is pumped through the centers of the capillaries, and cells are added to the outer chamber surrounding the bundle of fibers. The cells attach and grow on the outside of the capillary fibers, fed by diffusion from the perfusate , and can reach tissuelike cell densities. It’s an ideal system for studying the synthesis and release of biopharmaceuticals and are now being exploited on a semi-industrial scale Eg , in cultures, choriocarcinoma cells release more human chorionic gonadotrophin than they would in conventional monolayer culture and colonic carcinoma cells produce elevated levels of CEA

9. Spheroids The re- associaton of dissociated cultured cells leads to the formation of cluster of cells called spheroids S imilar to the reassembling of embryonic cells into specialized structures Principle: spheroid cultures is that the cells in heterotypic or homotypic aggregates have the ability to sort themselves out and form groups which form tissue like architecture The 3-D structure of spheroids allows the experimental study aspects of drug penetration and resistance to radiation or chemotherapy that are dependent on intercellular contact .

10. Contd … T he role of 3-D spatial configurations in gene expression in cell populations and the assessment of cytotoxic treatment. Treatment end points include growth delay, determination of the proportion of spheroids cured by treatment, and colony formation in monolayer after disaggregation of treated spheroids U se of spheroids to study the penetration of cytotoxic drugs, antibodies, or other molecules used in targeted therapy A pplication that is not possible in single-cell suspensions or monolayer cultures. Spheroids have proved useful in the study of cell killing by biologically targeted radionuclides

11. Rotating chamber systems Miniperm bioreactor: A conventional roller bottle in two-compartment chambers. T his kind of design is the MiniPERM TM , a two compartment cylinder with cells in the smaller compartment and medium in the larger, separated by a semipermeable membrane. It is rotated to ensure mixing, and the medium can be sparged or replaced without disturbing the cells or product. E.g ., monoclonal antibodies, the geometry of the chamber and the slow rotation tend to favor aggregate formation and enhance product formation.

12. Rotatory cell culture system(RCCS ) This bioreactor is available from Synthecon , Inc., as a disposable RCCS unit, or a reusable STLV (slow turning lateral vessel). Cylindrical vessel of bioreactor is completely filled with cell suspension, and then continuously rotated to maintain cells in a free fall state. Very low shear stress forces Minimal contact with vessel wall Stimulated zero gravity Quick production of speroids Culture multiple cell types Produce more differentiated complex epithelial shape Intrigued by the concept of growing cells in microgravity, in the 1980s NASA Gas exchange occurs from the cell-containing cylinder through a central silicone membrane When the rotation stops, the aggregates sediment and the medium can be replaced . To determine the effects of microgravity on cells with application to the space program this culture vessel has also provided a suitable bioreactor for bulk culture of tissue engineering constructs

13. Immobilization of living cells in alginate C omposed of alternating molecules of M and G, and divalent cations bind strongly between separate G blocks and initiate the formation of an extended alginate gel network. At present, numerous cell types can be genetically engineered to produce specific proteins of choice. By encapsulating such cells in alginate, a valuable vehicle is obtained for delivering specific recombinant proteins to the organism . . Thus, such alginate ‘‘bioreactors’’ may have an important therapeutic potential for the treatment of a number of diseases, in which the alginate may prevent the encapsulated cells from being destroyed by the immune system.

14. Filter Well Inserts Filter well inserts are a commercialization of a filter-based culture system the origins of which go back to the 1950s and used in various forms since then . A filter substrate provides an environment for studying cell interaction, stratification, polarization, and tissue modeling Polarity and functional integrity can be established as in thyroid ,intestinal and kidney, epithelium. Filter cultures allow generation of stratified epidermis They have used them to study invasion by granulocytes or malignant cells One of the major advantages of filter well inserts is that they allow the recombination of cells at very high, tissuelike densities, with ready access to medium and gas exchange, but in a multireplicate form .

15. Filter well inserts available in a variety of translucent or transparent materials, including polycarbonate, PTFE, and polyethylene teraphthalate , and ranging in size from 6.5 mm to 9 cm, suitable for 24-well, 12-well, and 6-well plates, or larger dish Filters can be obtained precoated with collagen, laminin , fibronectin , or Matrigels .

16. Cultures of Neuronal Aggregates Aggregating cultures of fetal brain cells have been extensively used to study neural cell differentiation The aggregating cells follow the same developmental sequence as observed in vivo , leading to an organoid structure consisting of mature neurons, astrocytes, and oligodendrocytes . A prominent neuropil is also formed In tumor biology, the aggregates can be used to study brain tumor cell invasion in vitro

17. Tissue Equivalents and Tissue Engineering

18. Advatages and disadvantages Advantages Development of a cell line over several generations Scale-up is possible Absolute control of physical environment Homogeneity of sample Less compound needed then in animal models Disadvantages Cells may lose some differentiated characteristics Hard to maintain Only grow small amount of tissue at high cost Dedifferentiation Instability, aneuploidy

19. References R.Ian Freshney ,(2005), Culture Of Animal Cells: A Manual Of Bacis Techniques , 5 th edition,Jhon Wiley & Sons,New Jersey,pp 436-450 R.Ian Freshney ,(2005 ), Culture Of Animal Cells: A Manual Of Bacis Techniques , 6 th edition,Jhon Wiley & Sons,New Jersey,pp 486-492

20. Gel Gel and sponge technique-