Rilianawati R1, Ago H2, Subintoro1, Elrade R1 , Kurnia A1 1 Agency for the Assessment and Application of Technology, Indonesia 2 Christian University of Indonesia, Indonesia
Introduction
Mesenchymal stem cells (MSCs) are a potential object for use in cell therapy and intensively studied by many research groups. MSCs are mainly characterized by the expression of surface markers and the potential for differentiation. MSCs express a series of specific markers (CD44, CD90, CD105, CD13, etc.) and should differentiate into cells of mesodermal origin such as adipocytes, osteoblasts, and chondroicytes. Due to the easily accessible anatomical location and the abundance of subcutaneous adipose tissue, the ADSC has the advantage of a simple and especially less invasive harvesting technique. The consideration of adipose tissue as a source of mesenchymal stem cells (MSC) for the engineering of autologous tissue is due to the fact that they are readily available in abundant quantities through minimally invasive procedures, as well as easy to grow and to multiply. It is possible to proliferate and differentiate in the desired direction of the network. Stem cell growth requires growth conditions such as optimal growth conditions such as ambient temperature of 37 ° C and a concentration of 5% CO2. Maintaining MSCs also requires a subculture process, that is, the process of moving MSCs from a complete culture medium to new media; a continuous subculture process can cause changes in MSCs. Stem cell viability can be disrupted by micro-conditions in wounds such as hypoxia, oxidative stress and inflammation. Alginate is an anionic polymer compound which can be obtained from brown algae and Pseudomonas and Azotobacter microbes. The alginate comprises a group of unbranched and non-recurrent exopolisakarida composed of two monomers, namely β-D-mannuronic acid and α-L-guluronic acid. The alginate will interact with divalent cations, such as Ca2 +, and form a 3D structure. The ability of alginate to form a 3D structure and its low toxicity characteristics and high biocompatibility make alginate widely used for the encapsulation process. Cell encapsulation is a method of cell capture in a semi-permeable polymer membrane, usually biocompatible encapsulation materials such as chitosan, hyaluronic acid, and alginate. Encapsulation is usually done to protect the cell. It is reported that the encryption of embryonic stem cells with alginate maintains the viability of stem cells for 110 days without undergoing differentiation and without requiring maintenance in the subculture. Encapsulating MSCs with alginate-CaCl2 is known to maintain viability under hypothermic conditions and maintain the viability of stem cells at different temperatures using a certain concentration of alginate and CaCl2 as a formula.
Material and Methods
Culture of mesenchymal stem cells (MSC)
The MSCs were cultured on α-MEM media taking into account the addition of platelet-rich plasma (PRP), penicillin / streptomycin and heparin. The replacement of the supports increases every 3 days. The MSC subculture is well growing when the MSCs are confluent ± 80%.
Optimization of MSC encapsulation
Optimization of CaCl2 concentrations: an alginate solution (Sigma Aldrich, medium viscosity) was prepared at concentrations of 1.25% (w / v). The 1 × 106 mesenchymal stem cells are mixed in 1 ml of alginate solution. The solution was then deposited in the form of droplets by extrusion method using a 31 G syringe with 200 mM CaCl2 solution with a contact time of + 30 min. The microcapsules formed were then washed using 0.9% NaCl 3 times and stored in a culture medium. The CaCl2 concentration to be used in the next step was selected based on the results of the encapsulated MSC viability test on days 0, 1, 7, 14, 21, 28.
Optimization of the alginate encapsulation: an alginate solution was prepared at concentrations of 1% in Aqua Sterile. The 1 × 106 mesenchyme stem cells are mixed in 1 ml of alginate solution. An alginate solution at various concentrations was then deposited in the form of droplets by extrusion method using a 31 G syringe to a CaCl2 solution with a concentration based on the result of optimizing the concentration of CaCl2 with a contact time + 30 min. The microcapsules formed were then washed using 0.9% NaCl and stored in a culture medium. These microcapsules are incubated at different temperatures at 4 ° C (refrigerator) and 37 ° C (incubator) for 0-28 days. The optimal concentration of alginate is selected by testing the viability and stability of the encapsulated MSCs.
Conclusion
The viability of mesenchymal stem cells in alginate microcapsules can last up to 28 days and there is no differentiation into adipose, chondrocytes, and osteoblasts. This may suggest that the encapsulated MSCs can be used to transport MSCs from one laboratory to another for more than 24 hours. And also due to the condition in another laboratory/clinic which will use MSCs do not always have liquid nitrogen storage. This result also shows that the alginate at low concentration and 100 mM CaCl2 is good for the growth of MSCs (as in the MTT result shown). This may be due to the fact that the encapsulated MSCs can adapt and develop in the alginate microcapsule at low concentration. In addition, the medium may also be easier to penetrate into the alginate of the microcapsule. However, these results require further investigation.
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