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.
CONCLUSIONS
1. 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).
2. 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
1. Optimal protocol of isolation, culture, differentiation mesenchymal
stem cells towards osteoblast from human bone marrow.
2. Apply autologous grafting mesenchymal stem cell differentiation
osteoblast for patient, who graft bone tissue and bone disease.
1
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:
1. Isolation, identification and culture successfully mesenchymal
stem cell sample from rabbit bone marrow.
2. Differentation mesenchymal stem cell towards the osteoblast
and cryopreservation cell after differentiation.
2
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, 4 chapters, 11 tables, 45 figures,
references: 9 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.
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.
23
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 3 week. There results were demonstrated histologic image. Afetr 6
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 1 hour, -800C for 1-2 days and -1960C for 2 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.
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.
3
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
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%
21
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 9 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 5 -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
5
DMSO and 90% FBS. After storage for 8 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
106 cell/ml. Cryovials were frozen and sequentially stored with cooling
rate 10C/min to -800C for 2 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 4 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, 4 week and 12 week groups.
CHAPTER 2. 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