Division of Tissue Culture

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Tissue Culture lab offers in vitro cell culture testing services for regulatory requirements and research. Main areas of interest are in vitro tissue constructs, stem cells and regenerative biology, liver and corneal tissue engineering. Involved in academic programs of the Institute such as Ph.D, MPhil and M.Tech.


Tissue Culture Lab of Division of Implant Biology maintains mammalian cell culture facility to meet the requirements of ISO standards. The laboratory offers in vitro cytotoxicity testing of biomaterials accredited by Le Comite Francais d'Acreditation (COFRAC) of France. In addition, laboratory also performs a wide spectrum of in vitro tests for general and specific cytocompatibility evaluation.


Current research focuses on development of in vitro tissues for regenerative medicine as well as organotypical models. Laboratory is well equipped with advanced tissue culture facilities and has more than two decades of experience and specialized in the isolation, culture and maintenance of variety of primary cells such as Hepatocytes, Liver Sinusoidal Endothelial Cells, Human Umbilical Vein Endothelial Cells, Bone Marrow Mesenchymal Stem Cells, Liver Progenitor Cells, Limbal Stem cells, Corneal Endothelial Cells and Skin Keratinocytes.

Tissue Culture lab prioritizes the research area to two main stream aspects - Corneal Tissue Engineering and Liver Tissue Engineering.

Intact in vitro tissues are produced using thermoresponsive culture surface without any enzyme treatment. This method well known as cells sheet engineering has been reported for the first time in India using in-house developed methods. New formulations of thermoresponsive polymers are continuously studied to expand the applications of bioengineered functional artificial tissue structures for tissue engineering and regenerative medicine. Bioengineered corneal cell constructs has been successfully transplanted to rabbit limbal stem cell deficiency model.

Tissue culture lab in collaboration with Division of Artificial and Internal Organs initiated development of indigenous bioreactor to act as bioartificial liver model. Studies are on going to develop appropriate procedures for isolation, harvesting and maintenance of hepatocytes with functional specificity and longevity.

The laboratory is also engaged in stem cell studies within the scope of prioritized research area. The stem cell research was initiated with ex vivo expanded limbal stem cells and development of cell sheet construct. Currently a variety of alternate stem cell sources are brought to the pursuit. Differentiation of liver progenitor cells under in vitro conditions is also examined to understand more about stem cells and their niche in expressing liver functions. Studies also focus on finding cell based therapeutics towards regenerating liver failure.

Extensive tissue damage or loss demands biological substitutes developed using functional cells and suitable scaffold. Various biological and synthetic materials are studied to enable the cells to perform as in the natural extra cellular matrices. Strategies are being formulated to improve differentiated functions in vitro and create functional three dimensional tissues.

Mechanotransduction is another important phenomenon that plays important role in the development of tissues. Cells receive physical signals from the environment and covert them into a biochemical response. Research is ongoing to study the effect of microgravity on hepatocytes and stem cells.

Offered to public

Cytotoxicity is a mandatory preliminary test to screen potential toxicity of biomaterials and medical devices before entering into advance testing and development. Tissue Culture Laboratory is equipped with cell culture facility which has the capability of testing cytotoxicity and cytocompatibility evaluation of biomedical materials as per ISO standards. The lab offers three basic tests according to ISO 10993-5 to assess the in vitro cytotoxicity of medical devices, viz. Test on extract, Direct Contact and Indirect Contact Test. Established mammalian cell lines are used to evaluate cytotoxicity of materials. General and specific cytocompatibility studies are also undertaken .

Test on Extracts

An extract of test material is obtained by incubating in Minimum Essential Medium for 24 h. The extract will be then placed on cell monolayer for a minimum duration of 24 h. Cytotoxicity will be determined by either qualitative or quantitative means. In qualitative evaluation the cells are examined for general morphology, vacuolization, detachment and cell lysis to determine a toxicity score. Quantitative evaluation measures cell viability and metabolic activity by estimating the number of cells, amount of protein, release of enzymes, release of vital dye, reduction of vital dye or any other measurable parameter. Reduction of cell viability by more than 30 % is considered a cytotoxic effect.

Direct Contact Test

The test material is placed in direct contact with cell monolayer for 24 h. The cytotoxicity is qualitatively evaluated by examining the monolayer microscopically for general morphology, cell density and cell lysis.

Test by Indirect Contact

In indirect contact test, cell monolayer is overlaid with agar before treatment with test material or its extract. After 24 h, cells are observed microscopically to determine cytotoxicity. A vital stain such as neutral red is added before or after the incubation with the specimen.

MTT Assay

The assay involves reduction of water soluble yellow MTT, tetrazolium salt to insoluble purple formazan crystals by metabolically active proliferating cells. The formazan crystals are solubilized and are quantified using an automated spectrophotometric multiwell plate reader. The ability of cells to reduce MTT indicates the metabolic activity which in turn may be interpreted as a measure of viability and cell number. L929 cells are seeded into 96-well plates and maintained in culture to form a semi-confluent monolayer. Cells are then exposed to extracts of test compound over a range of concentrations along with positive and negative controls. After 24 h exposure, cells will be exposed to culture medium containing MTT for 2 h and the formazan formation is determined for each treatment concentration. The ability to reduce MTT is compared to controls to assess the relative toxicity of materials.

Neutral Red Assay

The neutral red is a weak cationic supravital dye that penetrates cell membranes by non-ionic diffusion and accumulates in lysosomes. The neutral red uptake assay provides a quantitative estimation of uptake of neutral red by viable cells. Cells are seeded in 96-well culture plates and are treated with test materials for appropriate duration. Subsequently cells are exposed to medium containing neutral red for 2 h. Afterwards, internalized neutral red is extracted and quantified by obtaining absorbance using multiwell plate reader.

Cell Adhesion

Cell adhesion on biomaterial surface is a key factor in determining biocompatibility. Various physical and biological factors may influence cell adhesion on materials. Tissue culture lab offers cell adhesion test as a part of cytocompatibility evaluation. Cells seeded on materials and morphology will be evaluated under scanning electron microscope

Within Institute

All the services offered to public are applicable to within institute.


Tissue Culture Lab undertakes short term research projects specifically dealing with in vitro studies on topics coming under its scope.

  1. Nithya Joseph, Anil Kumar PR, Tilak Prasad, Leena Joseph, Sreenivasan.K, and Kumary TV, Intelligent Thermoresponsive Substrate from Modified Over Head Projection Sheet as a Tool for Construction and Support of In vitro Corneal Cell Sheet, Tissue Engineering Part C Methods, Accepted, August 2010.
  2. Viji, Mary Varghese; Prasad, Tilak; Kumary, TV; Optimization of culture conditions for an efficient xenofeeder free limbal cell culture system towards ocular surface regeneration Microscopy, Research & Technique. 2010, Available online
  3. Viji Mary Varghese, Vidya Raj; K Sreenivasan, TV Kumary,In vitro cytocompatibility evaluation of a thermoresponsive NIPAAm-MMA copolymeric surface using L929 cells. J Mater Sci: Mater Med 21:1631-1639, 2010
  4. Nithya, Joseph; Tilak, Prasad; Vidya, Raj; Anil Kumar, PR; Sreenivasan, K; Kumary, T.V. A cytocompatible poly (N-isopropylacrylamide-glycidylmethacrylate) coated surfaces as new substrate for corneal tissue engineering. JBCP 25: 58-74, 2010.
  5. Thomas N Abraham , Vidya Raj , Tilak Prasad , Anil Kumar PR, Sreenivasan K, Kumary TV. A novel thermoresponsive graft copolymer containingphosphorylated HEMA for generating detachable cell layers. J Appl Polym Sci 115: 52-62, 2010
  6. Sailaja G.S., Mohanty.M, Mohanan.P.V, KumaryT.V, Ramesh P and Varma H.K. Surface-Phosphorylated Copolymer Promotes Direct Bone Bonding. Tissue Engineering Part A. 15(10): 3061-3069, 2009.
  7. Raquel Magalhales*, Anil Kumar PR*, FengWen, Xuying Zhao, Hanry Yu, Lilia L. Kuleshova, The use of vitrification to preserve primary rat hepatocyte monolayer on collagen coated poly(ethylene-terephthalate) surfaces for a hybrid liver support system, Biomaterials 2009; 30(25): 4136-42. (*Equal contribution)
  8. Sailaja G.S., Sreenivasan, K, Yokogawab Y., KumaryT.V, Varma H.K. Bioinspired mineralization and cell adhesion on surface functionalized poly(vinyl alcohol) films . Acta Biomaterialia 5(5): 1647-1655, 2009.
  9. Kumary T.V, Sudip K.Ghosh,. Kundu S. C Characterization of fibroin and PEG- blended fibroin matrices for in vitro adhesion and proliferation of osteoblasts. Chitrangada Acharya, Journal of Biomaterials Science.- Polymer Edition 20: 543-565, 2009.
  10. Anil Kumar PR, Sreenivasan K and Kumary TV, An alternate method for grafting thermoresponsive polymer for transferring in vitro cell sheet structures - J Appl Polym Sci 105: 2245-2251, 2007.
  1. Process for chemical modification of cell culture substrate. Indian patent submitted on 2003.
  2. Method for cellularization of scaffolds using in vitro cell sheet constructs. Indian patent submitted on 2005.
  3. A temperature sensitive cell culture kit to generate, harvest and transfer in vitro tissue constructs for tissue regeneration. Indian patent submitted on 2009.