Research and Development

1. Transdifferentiation protocols for generating hepatocytes

In clinical studies, bioartificial liver (BAL) support has been performed with porcine hepatocytes (Watanabe 1997), immortalised human cell lines (Sussman 1994), and primary human hepatocytes to treat inborn errors of metabolism (Fox 1998). The availability and use of adult human Hepatocytes for clinical use is limited due to difficulties in maintaining primary Hepatocytes in culture. Several investigators have shown that human stem cells (embryonic, bone marrow, mesenchymal) can be differentiated into hepatocyte like cells in vitro (Sancho-Bru et al., 2009; Yan, Y et al., 2009; Banas, A. et al., 2009). The expression of albumin, cytokeratin 8, anti-alpha trypsin, glycogen, albumin and urea is positive in such differentiated cells. However, either transdifferentiation or dedifferentiation of such cells in artificial liver support system can be guaranteed. Functional capacity of adult Hepatocytes is much more complicated than these few parameters.Therefore, in the present project we plan to generate hepatocytes in vitro, from placental derived epithelial cells by mimicking in vivo hepatic microenvironment. This will be achieved through the addition of exogenous substances that can provoke normal metabolic processes such as oxidation, reduction, hydrolysis, and conjugation, influencing its ability to metabolize, detoxify, and excrete xenobiotics.

2. Use of amniotic membranes for generation of skin

The skin is the largest organ in the human body. Skin is made up of two layers, the epidermis and the dermis. Traditional ways of dealing with large losses of skin have been to use skin grafts but it gets rejected most of the time, but in future, use of amniotic membrane for generation of skin may be possible. Decellurized amniotic membrane was preserved and used for further process, preserved skin cassette was retrieved in water bath. Here we used PBS with antibiotics. PRP lysets were used for spreading on amniotic membrane. PRP coating of amniotic membrane was necessary to check histological studies. Media was changed after every third day in laminar air flow cabinet. This technology may act as milestone in skin regeneration techniques.

3. Decellularization and recellularization protocol for liver tissue engineering

Decellularization is proving to be a versatile technology in tissue engineering. Decellularized tissues and organs can be used in a variety of tissue engineering/regenerative medicine applications. Decellularization protocol can be considered an effective removal of cells and antigenic epitopes associated with cell membranes and intracellular components (HLA antigen).
These decellularized matrices can be used for the recellularization with the patient's own cells or different types of cells. The approach will provide an alternative tool to treat different diseases, organ failure or injuries. The source of material to get decellularized matrix will be from the pig. Decellularized matrix can be cryopreserved for the future use. Cord blood derived stem cells, cord tissue and placenta derived stem cells can be used for hepatogenic differentiation (Seung Hyun Hong et.al, 2005). These differentiated cells can be used for recellurization of generated DLM bio scaffold. The use of bioartificial liver support systems is further complicated and limited by the shortage of human hepatocytes. Human mesenchymal stem cells can be differentiated into hepatocyte like cells. (Kuan-Der Lee et. al, 2004).

4. Tissue engineering of Trachea

Trachea can be damaged due to accidents, cancer, autoimmune disorders. Decellularized tissues and organs can be used in a variety of tissue engineering/regenerative medicine applications. Decellularization protocol can be considered for an effective removal of cells and antigenic epitopes associated with cell membranes and intracellular components (HLA antigen). These decellularized matrices can be used for the re-cellularization with the patient's own cells or different types of cells. The source of material to get decellularized matrix will be from the pig. Decellularized matrix can be cryopreserved for the future use. After recellularization of matrix the histological studies will be carried out such as Eosin Haematoxylin staining. To achieve making a normal trachea two types of cells will be seeded on the trachea. Mesenchymal cells will be seeded on the outer side and epithelial cells will be seeded on the inner side of trachea. Organ transplantation is currently the only curative therapy for end-stage organ failure or diseases. It is evident that the choice of scaffold is crucial to enable the cells to behave in the required manner to produce tissues and organs of the desired shape and size and functioning ability.