Comparison and Optimization: Different Medium and a Novel Scheme for Rabbit Bone Marrow Mesenchymal Stem Cells Culture

in TEB, which might provide a novel optimizational scheme for rBMSCs culture.


Introduction
Large segmental bone defect is a tough problem for clinical treatment [1,2].Tissue engineering bone (TEB) transplantation provides a promising treatment for large segmental bone defect [2,3], its effect of promoting bone defect repair has been initially con rmed in basic experiments and clinical studies [4,5].The biological properties of seed cells are critical to the performance of TEB [6].Bone marrow mesenchymal stem cells (BMSCs) are a kind of pluripotent stem cells which derived from the early developmental mesoderm and stably present in bone marrow [7][8][9][10].Due to their good self-renewal ability and multi-directional differentiation potential, coupled with their weak immunogenicity, BMSCs are widely used as biological tissue engineering seed cells for bone and soft tissue repair [11,12].New Zealand rabbits are commonly used as experimental animals for bone and soft tissue injury and repair [13,14].
Although there are many reports on rBMSCs in recent years, there is still no consensus on the choice of medium for rBMSCs culture [15,16].
The physiological characteristics of BMSCs are strongly dependent on their culture environment [17].DMEM/F12, DMEM/Low glucose and DMEM/High glucose are the commonly used rBMSCs culture media [15,16], while the effect of different medium on the physiological characteristics of rBMSCs is still not clear and the optimal culture medium for rBMSCs is also uncertain.The proliferative capacity, multidirectional differentiation potential, adhesion ability and survivability in tissue engineering scaffolds of seed cells play an important role in the repair and reconstruction of damaged tissues [11,18].This study focused on the proliferation capacity, multi-directional differentiation potential, adhesion ability and vitality in TEB of rBMSCs cultured in different media.Further more, this study tried to optimize the culture mode of rBMSCs based on these results and nd a new effective rBMSCs culture method, which facilitated the culture of rBMSCs in vitro.

Experimental animals
A total of 5 healthy male New Zealand rabbits (aged 4~6 weeks, weight 400~800 g) were obtained from the Animal Experiment Center of Xiamen University.All rabbits were in good condition and had no related diseases based on examination.

Isolation and culture of rBMSCs
The 5-week-old male New Zealand rabbits were sacri ced with an intraperitoneal injection of an excess of 10% chloral hydrate (Sinopharm Chemical Reagent, China) and the hindlimbs of the rabbit were removed and immersed in 75% alcohol for 3 minutes.Then the muscles of femur and tibia were removed and the bone cavity was washed by PBS mixed with an appropriate amount of heparin sodium (0.04 mg/mL, Beijing Solarbio Science & Technology, China) for bone marrow.The suspension was collected and adjusted to the cell concentration of 2×10 8 to 1×10 9 /mL.A total of 5 mL rabbit mesenchymal cell separation uid (Tianjin HaoYang HuaKe Biological Technology, China) was added to a 15 mL centrifuge tube, and then the single cell suspension was placed onto the separation liquid level, at 450g centrifugation for 20 minutes.Cells in the second layer were collected in a Petri dish containing MSC complete medium [DMEM/F12 (HyClone, USA, the glucose concentration of 16.67 mmol/L) for F12 group or DMEM/Low Glucose (HyClone, USA, the glucose concentration of 5.56 mmol/L) for LG group or DMEM/High Glucose (HyClone, USA, the glucose concentration of 25 mmol/L) for HG group + 10% FBS (Biological Industries, Israel) + 1% streptomycin/penicillin (HyClone, USA) + 50 μmol/L β-mercaptoethanol (Beijing Solarbio Science & Technology, China)].The petri dish was placed in a cell culture incubator at 37°C with 5% CO 2 for 3 days, and then the medium was changed every 2 days.
As for the optimizational culture method (HG-LG) group, rBMSCs were isolated and cultured with DMEM/High glucose for the fomer 6 days, then they were digested with trypsin and cultured with DMEM/Low glucose.

Detection of MSC surface markers
The 3 rd passage cells of the 4 groups were collected respectively, then CD45 (Acris GmbH, Germany), CD44 (BD Biosciences, USA), CD34 (Thermo Fisher, USA), CD29 (Millipore Corporation, USA), goat antimouse secondary antibody (Multi Sciences, China) were used to detect their MSC surface markers according to the instructions [19].Fluorescence activated cell identify was performed with ow cytometry (Beckman USA) and data were analyzed with CytExpert (Tree Star, Ashland, OR, USA).

Multi-directional differentiation potential of rBMSCs
Osteogenic induction: The 4 groups of rBMSCs were seeded in 6 cm dishes with 3×10 5 in each one and cul tured at 37°C with 5% CO 2 for 24 hours.The original medium was then discarded and replaced for osteogenic induction with one containing 10 -2 mol/L of β sodium glycerophosphate (Dalian Meilun Biotech, China), 10 -8 mol/L of dexamethasone (Dalian Meilun Biotech, China), and 3×10 -4 mol/L vitamin C (Dalian Meilun Biotech, China).The medium was changed every 2 days for 2 weeks.Then calcium nodules in different groups were detected by Alizarin Red staining (Beijing Leagene Biotech, China) according to the instructions.Chondrogenic induction: The 4 groups of rBMSCs were seeded in 6 cm dishes with 3×10 5 in each one and cul tured at 37°C with 5% CO 2 for 24 hours.The original medium was then discarded and replaced for chondrogenic induction with one containing 10 μg/L of rabbit TGF-β1 (Dalian Meilun Biotech, China), 10 -7 mol/L of dexamethasone, 5×10 -5 mol/L vitamin C, 6.25×10 -3 g/L of insulin.The medium was changed every 2 days for 2-3 weeks.The induction was terminated and stained with Alison Blue Dye (Beijing Leagene Biotech, China) according to the instructions.Adipogenic induction: The 4 groups of rBMSCs were seeded in 6 cm dishes with 3×10 5 in each one and cul tured at 37°C with 5% CO 2 for 24 hours.The original medium was then discarded and replaced for adipogenic induction with adipogenic induction medium A containing 2×10 -4 mol/L of indomethacin, 10 -6 mol/L of dexamethasone, 5×10 -4 mol/L of 3-isobutyl-1-methylxanthine (China National Pharmaceutical Group Chemical Reagent, China) and 10 -2 g/L insulin (Dalian Meilun Biotech, China), and changed to adipogenic induction medium B containing 10 -2 g/L insulin after 2 days, and after 1 day, it was replaced with liquid A, and it was repeated.After 2 weeks, the induction was terminated and stained with Oil Red O (Beijing Leagene Biotech, China) according to the instructions.
The positive rate of dyeing (PRD) was calculated and compared by Image Pro Plus software.

Detection of cell proliferation ability
The cells to be detected were seeded in well plates with 2×10 3 cells/well, and then a total of 10 μL CCK-8 (Transgen Biotech, Beijing, China) reagents were added into each well and incu bated at 37°C with 5% CO 2 for 2 hours after culture for 1, 2, 3 and 4 days, the absorbance at 450 nm (A450) were determined at wave length of 450 nm with Microplate Reader (Bio-Rad, USA) according to the instructions.

Introduction of green uorescent protein (GFP)
Seed cells were labeled with GFP to monitor the survival of seed cells in TEB scaffold, this was inspired from the methods reported by Yuriko Kakimoto [21].10 5 primary rBMSCs were put into a 10 cm culture dishes with 8 mL culture medium, the lentivirus with GFP gene were put into each dish.The multiplicity of infection was adjusted to 100 and the density of polybrene was adjusted to 5 μg/mL.The medium was changed after 24 hours and then changed every 2nd day.The uorescence expression of cells were observed by an inverted uorescence microscope and these GFP-rBMSCs were collected and sorted by a Flow Separation Cell Meter (Beckman USA) and cultivated for ampli cation.

Preparation of deproteinized defatted cancellous bone scaffold (DDCBS)
Our research team has previously proved that DDCBS obtained from calf femoral heads had good porous structure, and the DDCBS implantation could effectively promote bone defect repair in New Zealand rabbits [22].In this study, the DDCBS obtained from calf femoral heads was used as scaffold for TEB construction.The cancellous bone of the calf femur was prepared into a cylindrical cancellous bone of 8 mm in diameter, deproteinized with 20% hydrogen peroxide at 37°C for 72 hours, and then degreased with ether at room temperature for 48 hours to prepare for DDCBS, as described before.Scanning electron microscope was used to observe the structure of the DDCBS (Fig 1).

Construction of TEB
The seed cells were adjusted to 3×10 5 /mL, 10 mL of cell suspension and a piece of TEB were put into a 15 mL centrifuge tube for mixed culture.The culture was carried out for 7 days with a rotary cell culture apparatus at 20 r/min (Sup.Fig 1).Then the TEB to be detected were placed into another 15 ml centrifuge tube and an appropriate amount of PBS was added to fully in ltrate the stent.The uorescence intensity of each group was observed and compared by living uorescence imaging system.

Statistical analysis
Results were expressed as mean ± standard deviation.Prism 5.0 (GraphPad Software Inc.) was used to make statistical analysis.Statistical comparisons for different groups were performed using one-way analysis of variance (ANOVA).P < 0.05 were considered statistically signi cant.

The morphology of rBMSCs in different culture groups
On the 6th day, the early primary rBMSCs cultured in the 3 media were fusiform, triangular and scattered There was no signi cant difference in the expression levels of each group (P>0.05)(Fig 4B).This indicated that the 3 rd passage rBMSCs of the 4 culture groups all had good MSC surface marker expression characteristics.

3.3
The multi-directional differentiation potential of the 3 rd passage rBMSCs in different culture groups Osteogenic, cartilage and adipogenic induction and staining experiments showed that the 3 rd passage of rBMSCs in the 4 groups all showed positive staining areas in Alizarin Red staining, Alison Blue and Oil Red O staining after induction (Fig 5A).This indicated that these 4 groups of cells all had certain osteogenesis, cartilage and adipogenic differentiation potential.Further analysis of each group of PRD showed that there was no signi cant difference in PRD between the 4 groups in the 3 staining experiments (P>0.05)(Fig 5B).The results indicated that the 3 rd passage rBMSCs cultured in different culture groups had similar multi-directional differentiation potential characteristics.

3.4
The proliferative capacity of the primary and the 3 rd passage rBMSCs in different culture groups CCK-8 cell proliferation assay showed that there was no signi cant difference among the A450 of primary rBMSCs in F12, LG, HG and HG-LG group from 0 to 24 hours (P>0.05).At the 48th hour, A450 of F12 group and HG group were higher than that of LG group (0.68±0.07 and 0.81±0.03vs 0.52±0.04), the differences were statistically signi cant.At the 72nd hour, A450 of F12 group and HG group were still higher than that of LG group (1.05±0.07 and 1.19±0.10vs 0.78±0.06), the differences were statistically signi cant.At the 96th hour, the A450 of HG group was higher than that of LG group (1.31±0.07vs 1.05±0.06)(P<0.05), while there was no signi cant difference in A450 between the other groups (P>0.05)(Fig 6A).There was no signi cant difference among the A450 of the 3 rd rBMSCs in F12, LG, HG and HG- LG group from 0 to 96 hours (P>0.05)(Fig 6B).This indicated that in the early stage, the proliferation capacity of rBMSCs in HG group was better than those of rBMSCs in LG group and F12 gorup, while in the latter stage, the proliferative capacity of rBMSCs in the 4 groups was similar.

The adhesion ability of the 3 rd passage rBMSCs in different culture groups
Cell adhesion experiment showed that the 75% cell adhesion time of the 4 groups from low to high was: LG and HG-LG group while higher than that of HG group, and the differences were statistically signi cant (P<0.01)(Fig 7).This indicated that the 3 rd passage rBMSCs' adhesion ability of LG group and HG-LG group had stronger adhesion ability than the other two, followed by F12 group and HG group.

The expression of GFP uorescence in the 3 rd passage rBMSCs of different culture groups
Inverted uorescence microscopy showed that the 3 rd passage rBMSCs in the 4 groups were highly expressed GFP uorescence.This suggested that the 3 rd passage rBMSCs of all 4 culture groups labeled with GFP uorescence could be used as tracer seed cells for TEB construction (Fig 8).

3.7
The vitality of the 3 rd passage rBMSCs in TEB in different culture groups After 1 week of TEB construction, the uorescence intensity of TEB in 4 groups were detected, and they were: F12 group (3.42±0.28)×10 .There was no signi cant difference in the uorescence intensity of TEB between the LG group and HG-LG group and there was no signi cant difference in the uorescence intensity of TEB between F12 group and HG group.However, the uorescence intensity of the former two groups was stronger than that of the latter two, and the differences were statistically signi cant (P<0.05)(Fig 9B).The results were consistent with the results of cell adhesion experiment.This further indicated that rBMSCs of LG group and HG-LG group had better vitality in TEB than HG group and F12 group.

Discussion
This study compared the physiological characteristics of rBMSCs cultured with DMEM/F12, DMEM/Low glucose, DMEM/High glucose and DMEM/High glucose for the rst 6 days and then DMEM/Low glucose.It was found that the rBMSCs in different culture groups all had good MSC characteristics, while the primary rBMSCs in DMEM/High glucose group had the highest proliferation rate and the 3 rd passage rBMSCs in DMEM/Low glucose group had the best adhesion ability and the strongest survivability in TEB.The rBMSCs cultured with DMEM/High glucose for the rst 6 days and then DMEM/Low glucose had good MSC characteristics, as well as strong proliferation ability of primary cells, strong adhesion ability and strong survivability in TEB of the 3 rd passage cells, which might provide a novel optimizational scheme for rBMSCs culture.
Sebastien et al compared physiological characteristics of human BMSCs cultured with DMEM/High glucose [23], DMEM/F12 and DMEM/Low glucose and found that the proliferation capacity of the primary BMSCs cultured with the 3 media from high to low were as follows: DMEM/High glucose group, DMEM/F12 group and DMEM/Low glucose group, while there was no signi cant difference in cell proliferation ability between the 3 rd passage of these 3 groups.In this study, the results were consistent with the results of Sebastien's and rBMSCs cultured with DMEM/High glucose with the glucose concentration of 25 mmol/L during the primary cell stage had the best proliferation ability.Gao Hai et al studied physiological characteristics of human BMSCs cultured with media containing different glucose concentration, and found that medium containing 10 mmol/L glucose promoted the proliferation of human BMSCs while high concentration of glucose (>30 mmol/L) inhibited the proliferation of human BMSCs [24].Although the optimal glucose concentration obtained by Gao Hai is different from that of Sebastien and that in this study, it is indicated that the glucose concentration in the medium had a great effect on the cell proliferation ability.
Studies have shown that medium with high glucose concentration within a certain range could effectively improve the proliferation ability of MSCs [23,24].However, some studies also have shown that medium with high glucose might accelerate cell senescence and apoptosis in seveal pathways [25][26][27].Yanan Kong et al cultured MSCs with high glucose (33 mmol/L) medium, and found that the apoptosis rate of cells remained low after culturing for 1 day, while the apoptosis rate of cells increased greatly after culturing for 5 days [25].And they came to the conclusion that cell apoptosis induced by high glucose was closely related to culture time.Tzu-Ching C et al cultured BMSCs with low glucose (5.5 mmol/L) and high glucose (25 mmol/L) medium respectively, they also found that high glucose could accelerate the senescence of BMSCs by promoting their ability of autophagy after culturing for 2 weeks [26].Yang Liu et al cultured nucleus pulposus derived MSCs (NPMSCs) with low glucose (5.5 mmol/L) and high glucose (25 mmol/L) medium respectively, and they found that high glucose medium cultured NPMSCs showed signi cantly decreased expression levels of stemness genes, related mRNA and protein, whereas increased expression levels of cell senescence markers and caspase-3 [27].Cramer et al applied medium with different concentration glucose (5.56mmol/L, 13.9mmol/L, 27.8mmol/L and 55.6mmol/L) to culture adipose derived stem cells (ADSCs) for 120 hours, and found that the hyperglycemia microenvironment could simultaneously reduce the proliferation ability of ADSCs and increase their apoptosis [28].In this study, the authors found that BMSCs reached 70-80% con uence on the 7th day and reached the standard of passage, meanwhile, changing the medium at this time point could avoid the adverse effects of long-term high glucose environment leading to apoptosis.Therefore, in this study, rBMSCs were isolated and cultured with DMEM/High glucose for the fomer 6 days, then they were digested and the medium was replaced with low sugar medium.
The results in this study showed that the 3 rd passage rBMSCs cultured with DMEM/F12, DMEM/Low glucose and DMEM/High glucose were all high expression in CD44 and CD29, while low expression in CD45 and CD34, and they had similar osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation potential.This indicated that rBMSCs cultured with DMEM/F12, DMEM/Low glucose and DMEM/High glucose all shared good MSC physiological characteristics.The results in this study were consistent with the results of Sebastien [23].Further more, the study found that in the 3 rd passage, rBMSCs in LG group had the strongest cell adhesion ability and the strongest survivability in TEB.The adhesion ability of seed cells is important for tissue engineering transplantation, and the stronger the seed cells' adhesion ability is, the easier the successful transplantation of TEB will be [28,29].Xue Xingying et al cultured human umbilical cord blood MSCs with DMEM/High glucose and DMEM/Low glucose, and found that the DMEM/Low glucose group had better adherence ability [30].This also indicated that different media have a effect on the adhesion ability of MSCs.Further, the study transplanted rBMSCs of different groups into DDCBS to observe the viability of these seed cells, and the results were also consistent with the experimental results above.
This study tried to optimize the culture protocol of rBMSCs: culturing rBMSCs with DMEM/High glucose for the rst 6 days and then DMEM/Low glucose.And the results showed that rBMSCs in HG-LG group concentrated strong proliferation ability of the primary cells of HG group, strong adhesion ability and strong survivability in TEB of the 3 rd passage cells of LG group, which might provide a novel optimizational scheme for rBMSCs culture.The physiological characteristics of BMSCs change at different growth stages, and so as to their environmental requirements, providing the corresponding medium according to the different growth stages of BMSCs might be the most bene cial scheme for the culture of rBMSCs.
The physiological characteristics of BMSCs are closely related to their culture environment.The presence or absence of serum [31,32], glucose content [24], related cytokines such as FGF [33], PH value [34] will all affect.Therefore, further investigations are required to develop a more satisfactory scheme for rBMSCs culture.

Conclusion
In summary, this study found that rBMSCs cultured with DMEM/F12, DMEM/Low glucose and DMEM/High glucose all had good MSC physiological characteristics, while the primary rBMSCs cultured with DMEM/High glucose showed the best proliferation ability and the 3 rd passage rBMSCs cultured with DMEM/Low glucose showed the best adhesion ability and the strongest survivability in TEB.RBMSCs cultured with DMEM/High glucose for the rst 6 days and then DMEM/Low glucose concentrated strong proliferative ability of the primary cells of HG group, strong adhesion ability and strong survivability in TEB of the 3 rd passage cells of LG group, which might provide a novel optimizational scheme for rBMSCs culture.
The morphology of the primary rBMSCs cultured in 3 different media in 2-6 days The primary rBMSCs in F12 group reached a fusion rate of 60% on the 6th day; the primary rBMSCs in LG group grew slower than the other two groups; the primary rBMSCs in HG group reached a fusion rate of 90% on the 6th day, which grew fastest.×60; n=5.
The morphology of the 3rd rBMSCs in 4 different cultured groups The 3rd passage rBMSCs of the 4 groups were all spindle-shaped or streamlined, closely arranged, while the morphology of rBMSCs in LG group and HG-LG group was more regular.×60; n=5.
The expression of stem cell surface markers in the 3rd passage rBMSCs of 4 different culture groups A: According to the ow cytometry results, CD44 (>95%) and CD29 (>90%) were prominently expressed, whereas CD45 (<10%) and CD34 (<5%) were barely expressed in the 3rd passage rBMSCs of different culture groups.Stained cells were represented in green, whereas unstained cells were in red.B: Statistical analysis of ow cytometry results.n=3.
The multi-directional differentiation potential of the 3rd passage rBMSCs in 4 different culture groups A: The 3rd passage rBMSCs in 4 different culture groups all showed positive staining areas in Alizarin Red, Alison Blue and Oil Red O staining after induction.The osteogenic induction staining was black, the chondrogenic induction staining was blue, and the adipogenic induction staining was red.×100.B: Statistical analysis of multi-directional differentiation induced staining results.n=3.
The proliferation ability of the primary and the 3rd rBMSCs cultured in different media A: The blue, red and green colors in the gure represented the A450 curves of the primary rBMSCs in F12 group, LG group and HG group, respectively.During the culture process, the proliferation ability of rBMSCs in HG group was the strongest, while the proliferation ability of rBMSCs in LG group was the weakest.The green asterisk refered to HG versus LG, and the blue asterisk refered to F12 versus LG.B: The blue, red, green and black colors in the gure represented the A450 curves of the 3rd rBMSCs in F12 group, LG group, HG group and HG-LG group, respectively.n=3; * P<0.05; ** P<0.01.
The adhesion ability of the 3rd passage rBMSCs in 4 different culture groups The blue, red, green and black colors in the gure represented the cell adherence rate curves of the 3rd passage rBMSCs in F12 group, LG group, HG group and HG-LG group, respectively.The 3rd passage rBMSCs in LG group and HG- LG group had the strongest adhesion ability, followed by DMEM/F12 group, and the adhesion ability of the 3rd passage rBMSCs in HG group was the weakest.n=3.
The expression of GFP uorescence of the 3rd passage rBMSCs in 4 different culture groups The 3rd passage rBMSCs in the 4 groups were highly expressed GFP uorescence after staining.×60; n=3.

Figures Figure 1
Figures Fig 2)while rBMSCs cultured in HG group grew fastest.The 3 rd passage of rBMSCs cultured in the 4 groups were spindle-shaped or streamlined, closely arranged, while the morphology of rBMSCs in LG group and HG-LG group was more streamlined (Fig3).