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Isolation, identification and culture successfully mesenchymal stem cell sample from rabbit bone marrow. Differentation mesenchymal stem cell towards the osteoblast and cryopreservation cell after differentiation.
24 4.2.3.Assessment of cell viability Most of the thawed cells can attach surface of culture flasks In addition to characteristics described above, another defining feature that thawed osteoblast still kept was their morphology These observations clearly showed cryopreserved osteoblast could be stored and maintained hight degrees of viability and differentiation potential With these results, we believed that cryopreserved osteoblast might become promising candidate cell source for cell –based therapy BACKGROUND Partial loss of a bone of the extremities due to trauma, tumor excision, bone disease, etc… presents as a substantial impediment throught the life of patients, and numerous efforts have been made to reconstruct the normal function of the skeletal system However, such efforts still have many limitations and problems When using bone graft, problems may develop in the donor area in general autologous bone graft and immunological problems while the spread of disease may also develop in allografts Despite advanced and optimized surgical procedures, approximately 5% fracture sustained annually in the United states fail to achieve bony union There may be faster patient recovery and an absence of these problems when stem cell are used Cell culture has large numbers Cell differtiation, they would be expected to become a useful cell source for treatment, has become more research favorable in the world and Vietnames Therefore, the maintenance of stem cell initial characteristics is crucial, and it may be possible to preserve them by satisfactory cryopreservation technologies This is expeccially significant because the supply of stem cells that are limited could be happed any time In fact, if long term cryopreserved stem cell still retained biological ability, they would be expected to become a useful source for regenerative medical progression Everyday, our laboratories we provide bone tissue for 10 patient to autograft and allograft Purpose bone tissue engineering conjugate with stem cell engineering that can regenerate bone tissue with an appropriate structure and function Therefore, we conducted “experimental research to apply protocol culture and cryopreservation of osteoblast from differentiation bone marrow mesenchymal stem cell” Objective: Isolation, identification and culture successfully mesenchymal stem cell sample from rabbit bone marrow Differentation mesenchymal stem cell towards the osteoblast and cryopreservation cell after differentiation CONCLUSIONS Isolation, identify and culture successfully mesenchymal stem cells from the rabbit bone marrow, thereby providing a summary process for important steps such as anesthesia, position, aspirated marrow technology, isolation, culture mesenchymal stem cell (identified as mesenchymal stem cell) Differentiation successful bone marrow mesenchymal stem cell into osteoblast After cell differentiation were identified by immunohistochemistry with antibody osteocalcin, staining with Alizarin red, observation of mineral crystal under scanning electron microscope The result confirm the differentiation mesenchymal stem cell into osteoblast Cryopreservation cell after differentiation by slow freezing, rapid thawing in freezing medium 10% DMSO, 15% FBS After thawing, viability rate over 80% Most of the thawed cells can attach surface of culture flasks, cell is still developing well, cell shape is unchanged RECOMMENDATIONS Optimal protocol of isolation, culture, differentiation mesenchymal stem cells towards osteoblast from human bone marrow Apply autologous grafting mesenchymal stem cell differentiation osteoblast for patient, who graft bone tissue and bone disease 2 23 The thesis is a new contribution to the field of stem cell research in Vietnam Especially successful differentiation of bone marrow mesenchymal stem cell into osteoblast with morder identify technology This is the first result in Vietnam This result has great practical significance in the treatment of bone and joint disease by stem cell therapy Through the thesis, the author and the stem cell research team of the Department of Histology and Embryology, Ha noi Medical University have mastered technology as the steps in a process, starting with the isolation technique, culture, differentiation, identify, cryopreservation cell We will build a cell bank that is differentiation according to treatment and research STRUCTURE OF THE THESIS The thesis consitts of 127 pages, chapters, 11 tables, 45 figures, references: Vietnamese: 116 foreign language Background: 02 pages; chapter1: overview 37 pages; chapter 2: Objects and Methods:18 pages; Chapter 3: results: 30 pages; Chapter 4: Discussion 37 pages; conclusions: 02 page Recommendations: 01 page; lists of related articles: references; appendix 4.1.3.8 Injection of autologous cultured osteoblast in experimental When we compare the results in the two groups studied the results showed that in the experimental, the time for new bone tissue regeneration and healing of bone quality was better and faster the control group at week There results were demonstrated histologic image Afetr weeks, the comparisons between the team were very difficult However, if based on SEM picture, the regeneration and repair in experimental were not similar, while the team for bone healing pretty good results, the results one the bone healing not as good as the control group 4.2.Cryopreserved osteoblast 4.2.1.Cell viability rate The slow freezing – rapid thawing method using dimethylsulfoxide (DMSO) as a cryoprotectant can be easly applied to the cryopreservation of cell such as osteoblast The cell are suspended in freezing medium containing 10% DMSO transferred into cryovials and then frozen by steps with slowly decreasing temperatures 40C for 10 min, -200C for hour, -800C for 1-2 days and -1960C for weeks In addition to keeping balance to protect cryopreservated cells, toxic and osmotic damage by decreasing temperature slowly with low concentration of cryoprotectants When slow freezing method cryoprotectant as replacement for intracellular water The viability of osteoblast thawed from different freezing media varied as well However the difference between viability rate of cryopreserved groups and freezing medium After thawing, cryopreserved cells had a fairly hight degree of viability, about 87% in MT3 freezing medium MT1 freezing medium was not suitable, the viability rates had low 61.28% Our study was suitable result of Verdanova M (2014) Cryopreserved osteoblast in differentce freezing medium the cells were frozen in freezing tubes at -800C at a cooling rate of 10C/min Frozen cells were transferred into liquid nitrogen 87% of frozen osteoblast survived 24h after thawing from the standard freezing medium 10% DMSO and 25% FBS CHAPTER 1: OVERVIEW 1.1 Mensenchymal stem cell 1.1.1 Character mesenchymal stem cell Friedenstein AJ (1976) were discovered stromal stem cell, Who showed that bone marrow contains a population of plastic- adherent highly proliferative cells, that were able to form colony of fibroblast (hence the name colony – forming unit – fibroblast, CFU-F) Mesenchymay stem cell (MSC) display multilineage differentiation potentials Invitro, MSC are capable of differentiation a long osteogenic, chondrogenic and adipogenic lineages Morphological MSC population are fusiform or stellate cell not easily distinguished from fibroblast In electron micrographs, they have a coarser chromatin pattern, fewer mitochondria and their sparse cytoplasm contains little or no endoplasmic reticulum 22 The osteogenic potential of the isolated mesenchymal cells was confirmed with the qualitative analysis based on the staining of the calcium accumulated in the extracellular matrix, which is one of the main events in the osteogenic differentiation process The evaluation time points 7-14 days the cell under osteogenic conditions exhibited morphological changes typical of the osteoblastic phenotype The observation of the major mineralization events after 30 day of culture is white crystals in accordance with previous reports for MSCs suggesting similar osteogenic potentials(Quiroz FG 2008) MSC were plated and treated for osteogenic differentiation as described To assess differentiation of rabbit bone marrow mesenchymal stem cells, some bone – specific markers were analyzed using histo and immunohisto- chemistry techniques in cells grown in osteogenic differentiation medium There aggregates were characterized by deposits of amorphous material The nodular aggregates in osteogenic cultures stained with alizarin red S, demonstrated that amorphous deposits observed under the microscope were actual calcium deposits Alizarin red – positive nodular aggregated were already present at day 15 The positive alizarin red were lager and stained intensively, indicating that a more extensive calcium deposition had occurred Control cultures showed only minimal background staining Under SEM appearance white crystals of mesenchymal stem cells differentiation into osteogenic line The cells were immunocytochemistry stained positive for osteocalcin Osteocalcin, a bone – specific glycoprotein that binds calcium and may promote calcification of the bone matrix has been used as a late marker for osteogenic differtiation (Karaoz, 2009) The bone marrow mesenchymal stem cells model used in this study has basal expression of the osteogenic markers, only cultures under osteogenic conditions showed osteocalcin marker for the induction of the osteogenic differentiation as evidenced though the morphological changes and the mineralization of the extracellular matrix However, MSC constitute only a small proportion of the cell in bone marrow 0,001 – 0,01% of total nucleated cells ̴ 1/10 HSC Now, MSC were isolated from various sourcer as umbilical cord blood, adipose tissue, bone marrow, cartilage… 1.1.2 Marker MSC (identified by the minimal criteria of the international society for cellular therapy Table 1: Marker for human mensenchymal stem cell Positive ≥ 95% Negative ≤ 2% CD105, CD73, CD90 CD45, CD34, CD14 or CD11b, CD79α or CD19, HLA-DR However, this study investigated whether there are marked differences in surface markers between rabbit and human MSC 1.1.3.BMMSC isolation and detection BMMSC were isolated from the marrow aspirate by gradient centrifugation, using Ficoll –Paque (Sigma, USA) The mononuclear on the top with an equal volume of Ficoll – Paque The isolated cells contains MSC, hematopoitic cells and monocytes The culture remove the hematopoietic cells and futher expanded MSC 1.2 Criteria to define MSC (ISCT) One notable characteristic is plastic – adherent ability of MSCs ,When they are cultured in appropriate media Another property MSCs must have differentiation potential into osteoblasts, adipocytes and chondroblasts in ni vitro culture with know stimulators Lastly, MSCs were confirmed with the phenotype that were positive for CD73, CD90, CD105 and negative for CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR 1.3 In vitro differentiation: MSCs are multipotent cells that can undergo multi –lineage differentiation MSCs were differentiation in to osteoblast, adipocytes, chondroblasts… using the standard induction media 4 21 1.4 Cryopreservation cells Cryopreservation cells sequentially stored with slowly decreasing temperatures Between -50C to approximately -150C, ice forms (either spontaneously or as result of artificially introducing ice crystals to the solution) in the external medium; however, the cell’s contents remain unfrozen As the cell increased solute concentrate and cryoprotectants (CPAs) in response to increasing extracellular osmolality It is clear from the cryobiology theory decribed above that whether the ability of water to move across the cell membrane agiven cell type depends cooling rate The subsequent physical events in the cells depend on the cooling rate If cells are cooled enough slowly intracellular solution maintain equilibrium with external medium However, if cells are cooled too rapidly than the water transport external medium as result introducing ice crystals intracellular cell, but morphological cells aren’t change Following storage, optimal cooling cell will lose water in the partialy enough to maintain equilibrium If the cell aren’t cooled rapidly, ice crystals froms in the extermal medium, they will experience a severe volume shrinkage In general, cryopreserved cells are equilibrium between losen water and cooling rate Role cryoprotectants All discussion above refer to the role of CPAs helps avoid the risk and possible damage caused by low temperature or frozen temperature CPAs is induce the cell water/cytoplasm to form a glass than to crystallize The CPAs suppress the salt concentration in solution; reduce harmful cell shrinkage at any given temperature In the presence of macromolecular compounds, a proportion of water does not crystallize during the freezing process Cryopresesation stem cell Xie XH (2012) Cryopreserved rabbit bone marrow mononucle approximately 107 cell/1ml re – suspended cryopreserved medium 10% expressed CD44, CD90 They also were negative for CD14,CD34 These results demonstrated that bone marrow MSCs, in this study, is relatively satisfactory to recommended minimal criteria of the mesenchymal and tissue sem cel committee of the international society for cellular therapy CD14, CD34 are common markers of circulating blood cells Among of them, marker Cd14 lipo- polysaccharide receptor, is a surface antigen of monocytes, macrophages and endothelial progenitor cells The CD34 protein, a surface glycoprotein, expressed on developmentally early hematopoietic stem cells and progenitor cell as well as endothelial cells It has been suggested that CD34 negative stem cell may generate hematopoietic stem cell Lee TC (2014) reported that the surface expression of markers differed between human and rabbit MSCs, Whereas the surface expression of 16 markers was he same in the two cell lines Analysis results also showed that rMSC were positive with markers CD44 (97.32%), CD90 (95.37%) and the rate of negative markers are CD14 (0.17%), CD34 (0.36%) 4.1.3.6 Adipogenic differentiation rMSC in the adipogenic differentiation experiments Although adipogenic differentiation potency of rabbit bone marrow MSC was examined for 10-15 days, the onset of phenotypic changes was noticed after -7 days of incubation During differentiation, cell shape began to transform into ovoid morphology and the lipid droplets showed a tendency to accumulate in the cell periphery At the beginning of the second week, the lipid droplets enlarged and invaded the entire cytoplasm like adipocyte differentiated rabbit bone marrow MSCs Control cells did not differentiate Oil red O staining positive adipocytes were red color which demonstrated the committed differentiation of MSC into adipocytes when culture in induced medium with know adipogenic factors 4.1.3.7.Osteogenic differentiation 20 marrow graft obtained after concentration cantained progenitor cells The use of percutaneous autologous bone marrow transplantation in nounion and a vascular necrosis of bone 4.1.3.4.Characterization of cell proliferation assay The proliferation of the cell lines was demonstrated by growth curves for the measurement of cell proliferation the growth curves test was performed The cell proliferation was using evaluation method effects of growth factor, hormone, nutrient medium, difference factor on cell line The cell growth or increases the number of cell is that work has been carried out on almost all aspects of mesenchymal stem cell biology Because they were determined effect difference factors: altering nutrition, mitosis factor… Liu C (2014) reported that Annulus fibrosus (AF) sample were isolated from intervertebral disc (IVD) of rabbits The cell was cultured in DMEM – LG supplemented with 20% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin Cell proliferation capacity was tested using cells at passage three though MTT assay The typical population doubling time was 17.8 hrs 4.1.3.5 Expression of surface markers Immunofluoresvence staining and flow cytometry analysis indicated that MSC at P4 were strongly positive CD44,CD90 The cells were negative for CD14, CD34 All markers are commonly used in analyses of MSCs These data coincide well with the reported data of mesenchymal stem cells characteristics Tan SL (2013) repored that both rMSC and hMSC at P2 or P3 were determined for phenotypic characterization using flow cytometry The cultured rMSC were positive CD90 (96,9%) and hMSC (100%) Our result were positive CD90 (95.37%) Lee TC (2014) studied that rMSC CD44 (97.32%) This result was suitable with studied of ours Characterization of bone marrow mesenchymal stem cells is also based on the expression of specific markers MSCs were strongly DMSO and 90% FBS After storage for weeks, cryopreserved cells had a fairly high degree of viability, about 96,49% Kotobuki N (2005) cryopreserved MSCs in medium: DMSO and FBS After storage for (0,3-37 months), the viability rates of cryopreserved groups were approximately 90% Both fresh and cryopreserved MSCs analysis their marker This result also revealed that the cryopresenvation did not influence the marker Zeisberger SM (2011) cryopreserved AT- MSC for a concentration 10 cell/ml Cryovials were frozen and sequentially stored with cooling rate 10C/min to -800C for days Immediately after wards, the cryovials were immersed in liquid nitrogen Following storage, cryopreserved cells were medium with diffence concentrate, DMSO will difference ice froms intracellular and extracellular solution Shimizu T (2013) cryopreserved osteogenic matrix cell sheets (OMCS), in which bone marrow - derived MSC were culture to confluence and differentiated the osteogenic lineage to form osteogenic matrix cell sheets (OMCS) OMCS are into cryovials containing cryopreservation medium (cell Banker 1®) Tube were then transferred to a freezer (-800C) and stored at -800C for or 12 weeks After thawing, the viability rate of cryopreserved groups were 63,3± 8.6%; 61.1 ± 6.5%, respectively After thawed OMSC are still capable of producing minerlzed There was no significant difference in ALP activing between the fresh, week and 12 week groups CHAPTER SUBJECTS AND METHODS 2.1 Subjects of the reseach Subjects reseach: Bone marrow mononuclear cells were isolated from the marrow Material reseach: Bone marrow solution Standard of rabbit: 30 rabbit, 6-8 week old male rabbit for 1.8-2.5kg 19 Total sample bone marrow :30 sample, culture 30 sample, cryopreservation 30 sample 2.2 Methods of the reseach 2.2.1 Reseach model Isolation, culture, identified and differentiation MSC from bone marrow Cryopreservation osteoblast then evaluation of viability rate and differentiation ability of cryopreserved groups 2.2.2 Content reaeach 2.2.2.1 Isolation, culture, identified MSC from BM and differentiation into osteoblast Aspirated BM: Choice of position bone marrow aspirate and total volume solution Isolationed MSC: Bone marrow mononuclear cells by gradient centrifugation Briefly, bone marrow was loaded on the top with an equal volume of Ficoll –Paque (sigma, USA) preloaded in a centrifuge tube and centrifuged at 3000rpm for 20 minutes in room temperature Culture: Primary culture: Bone marrow mononuclear cells were suspended in culture medium and seeded into flask The cells were cultured at 370C, 5% C02 The subsequent medium change was conducted at day intervals When these primary culture cells reached 70 – 80% confluence, they were sub cultured Second culture: These primary culture cells were harvested using Trypsin/EDTA and sub cultured at 1: Detection cell: Immunostaining cells: Cells were fixed with 4% PFA at room temperature for 10 and washed with PBS, followed by permeabilization with 0,2% Triton X100 for 10 and blockage with 4% goat serum Cells were incubated with primary for day 250C Subsequently, antibodies of CD14, CD34, Under the culture condition used in this study, rBm-MSC grew consistently for 10 Passages without loss of proliferative ability and significant changes in morphology 4.1.3.2 Second culture The cells, referred to stem cells, proliferated more rapidly than primary culture (in the first passage of culture), fibroblast – like cells reached 70 – 80% confluence at day -7, at which point these cells were suitable sub cultured These cells were detached by incubating with trypsin/EDTA solution Complete medium was added to inactivate the trypsin and sub cultured at a ratio of 1:3 Purification of mensenchymal stem cells candidates was achieved by removing nonadherent cells during sub sequent changes of medium When sub culture homogenous after passage three 4.1.3.3 Conony formation Such stem cells should possess clonogenicity, self – renewal capability and multipotency the common characteristics of mesenchymal stem cell To test the colony forming capability of mesenchymal stem cell, a colony forming unit – fibroblast (CFU-F) assay was performed Base on size, morphology of cell was knew progenitor cells Our study MSCs were culture in the growth medium supplemented with 10% FBS The number of fibroblast colony –forming mean 107 ± 25 Liu C (2014) to test the colony forming capability of rabbit AF – derived cells, they were cutured in the growth medium supplemented with 20% FBS The colony formation capacity was largely dependent on the initial cell seeding density An initial seeding of density of 200 cells/cm2 was found to result in the highest efficiency of colony formation To test the colony forming unit (CFU-F) as an indicator of stromal cell activity Results are expressed as the mean number of mesenchymal stem cell per one million mononucleated This technique, the number of mesenchymal stem cell that was transplanted was estimated by counting the fibroblast conoly forming units (Hernigou.PH 2005) The bone 18 adherent, their shape and size and their ability to help form colonies in culture Mensenchymal stem cell obtained by this method were adherent to plastic and demonstrated fibroblastic spindle shape as observed under microscope In present study, spindle – shaped fibroblast – like cells from bone marrow were purified by repeated medium chang at initial hours of culture and diminishing the trypsinization time Using the method described in this study frequent medium change may prevent adherence of many of the non – MSC and hematopoietic population to the culture dish In the study, The mean number of rMSC was determined in primary culture Phase 1: The mean number of adherent cell and proliferation was lower than Phase because the first reseach haven’t stardard protocol Secon reseach: The mean number MSC in P0 was 1.8 x104cell/106BMMNCs MSC candidate proliferated slowly in primary culture Our results was suitable for XieXH (2012) MSC in P0 was 1.92 x 104/106 BMMNCs Mesenchymal stem cells obtained by this method were relatively homogeneous in morphology rMSC characteristics similar to those of hMSCs based on the following observing under light microscope The mesenchymal stem cell are small fusiform or stellate cells In electron micrographs, they have a coarser chromatin pattern, fewer mitochondria and their sparse cytoplasm contains little no endoplasmic reticulum Although similar in their morphological appearance the rMSC cell length (202.66 ± 840µm) and width (13.09 ±0.91µm) were significantly longer and wider than hMSCs (152.04 ± 43.35µm) in length; 9.82 ±0.66µm in width (p