Selection of basal medium for culturing human umbilical cord mesenchymal stem cells in combination with human platelet lysate

Different basal media have very different effects on cell growth, proliferation and differentiation potential of cultured human umbilical cord mesenchymal stem cell (hUC-MSC). This issue has not gotten enough research. The goal of this study is to select high safety and low cost basal media for hUC-MSC, which performs best in combination with commercial human platelet lysate product for cell proliferation and differentiation functions, especially suitable for high-passage hUC-MSC cultures. Three different basal media were combined with 5% UltraGROGAdvanced to culture P0 to P8 passage hUC-MSC. Cell morphology, cell expansion ratio, proliferation, differentiation and cell surface markers were examined and analyzed in order to draw conclusions.


Background
Mesenchymal stem cells (MSCs) are a group of multipotent stem cells with the properties for self-renewal and multi-directional differentiation. When expanded in vitro, they are able to differentiate into many kinds of cells including chondrocytes, adipocytes, osteoblasts, nerve cells and muscles. MSCs generally positively express CD105, CD90, CD73, but do not express CD34, CD45 and other hematopoietic stem cell (HSC) markers. A large number of clinical trials are in progress, and several clinical studies have shown that MSCs plays a good role in the treatments of various diseases. Studies have shown that injection of MSCs can increase the molecular levels of IL-10, IL-37 and IL-17, and has better effects in improving ischemic myocardium [16]. MSCs have good therapeutic effects on nervous system injuries [1][2][3], musculoskeletal injuries [4], liver and lung diseases and dysfunctions [5], liver and lung brosis [6], immune system diseases, etc. MSCs also participate in the body's immune regulation and cell growth. All these results bring new hope for the treatment of many di cult diseases.
MSCs come from a wide range of sources. They were rst discovered in human bone marrow. Later, researchers successively isolated and them from fat, umbilical cord, placenta and other tissues. Umbilical In recent years, studies have shown that allogeneic platelet lysates are superior to FBS in culturing MSCs [5, 6, and 7]. MSCs cultured with platelet lysates have strong differentiation abilities and can maintain good cell morphological structures [8,9]. It was found that the immune functions of cells cultured in platelet lysate was lower than that of cells cultured in FBS [10,11].
This was supported with a report of 13 patient's treatment with allogeneic MSCs expanded using medium containing platelet lysate [12]. Recent data on the use of MSCs to treat GVHD showed that 77% of medical institutions use platelet lysate-containing media to expand MSCs (54% comes from the blood bank, 23% comes from commercial sources), while the rest 23% of the institutions still using FBS [13].
When bone marrow and fat-derived MSCs were cultured with media containing FBS, human serum ,or human platelet lysate,the report found that fat-derived MSCs cultured with platelet lysate have better initial growth, but lower later proliferative functions than those using FBS and human serum while bone marrow-derived MSCs grow best in platelet lysate culture system [14,15]. These studies did not distinguish the types of basal media used, so how basal media impact the cultivation of MSCs was unknown At present, the methods for cultivating hUC-MSC are by no means perfect. There are many kinds of basal media on the market. The formulation and ingredient quantities of each medium are different. There are differences in cell functions, cell recovery, cell purity, etc. So they are not all best suitable for hUC-MSC culture. The purpose of this study was to select a basal medium with good stability and low cost, when combined with animal-free platelet lysate, can ensure the high-passage hUC-MSC cultures of stability, proliferation, differentiation, immune-phenotype expression, and maintaining the MSC characteristics.
In this paper, umbilical cord tissue block adherence-migration plating method was used to isolate MSCs.
Three commonly used basal media with high performance and low cost were used for comparison. HUM-MSCs were cultured with MSCBM from Dakewe, α-MEM and IMDM from Hyclone, combined with platelet lysate product UltraGROGAdvanced respectively. Functional tests on cells of different passages were performed. Basal medium which is best suited for Low-cost large-scale cultivation of hUN-MSCs was chosen based on the indicators of cell morphology, growth cycle, growth status, cell number, cell proliferation function, cell surface phenotype expression, cell differentiation function and others.

Human Umbilical Cord Mesenchymal Stem Cell Culture Methods
In a biological safety cabinet, the umbilical cord is disinfected with 75% alcohol brie y, and then washed twice with physiological saline. In a dish, the cleaned umbilical cord was cut into sections of 2-3 cm in length, and the Wharton's jelly was peeled off. The Wharton's jelly was transferred into a centrifuge tube and was cut into 1-2 mm 3 blocks using surgical scissors. The tissue blocks were plated at 2 grams tissue per T75 bottle, cultured with different basal media plus 5% UltraGROGAdvanced, placed in a carbon dioxide incubator and incubate at 37 °C with 5% CO2. The media were change after 4-5 days. When reached 70% con uent or more, the cells were digested with 0.125% TrypLE to obtain single cells, and plated at 1 × 10 6 cells / T175 ask, cultured with the same medium as before. After the cells had reached 90% con uent, the cells were passaged again. The cells are passaged and cultured up to the 8th passage (P8) according to this method.
Immunoassay For Cellular Phenotypes P1, P3, P5, P8 hUC-MSCs were digested into single cells with 0.125% TrypLE, and collected by centrifugation. After washing twice with physiological saline and cell counting, 1 × 10 6 cells were incubated with CD105, CD34, CD45, CD73, and CD90 antibodies at 4 °C in the dark for more than 30 minutes. Washed three times with PBS, and the expression of immune-phenotypes were detected with ow cytometry, and a negative control was also set.

Detection Of Cell Proliferation Function In Different Passages
P1, P3, P5, P8 hUC-MSCs were digested into single cells with 0.125% TrypLE, and centrifuged to collect the cells. After washing twice with saline and cell counting, adjust the cell concentration to 1000 cells/ml and plate into 24-well plates at 0.5 ml/well and cultured again. 24hrs later, three wells from each condition were digested and counted. This was done at the same time every day for 8 days. Each

Detection Of Cell Differentiation Function
Osteogenic differentiation: P8 hUC-MSCs were digested into single cells with 0.125% TrypLE, and collect the cells by centrifugation. After washing twice with saline and cell counting, adjust the cell concentration in osteogenic differentiation medium to 1 × 10 4 cells/ml and plate into 24-well plates with cover slips at 1 × 10 4 cells/well. Setting up three parallel wells for each condition also set up one negative control well. The medium was changed the next day, and every 3 days afterward. On the 14th day in culture, the cells were rinsed gently with PBS once, xed for 30 minutes in 4% paraformaldehyde, stain with Alizarin S for 5 minutes, washed once, stained nuclei with hematoxylin for 1 minute, washed with water for 1 minute, dehydrated with gradient alcohol for two minutes, clari ed with Xylene for 5 minutes, and mounted with glycerin gelatin. Observed under the microscope and randomly selected 5 elds to count positively stained cells for statistical analysis.
Adipogenic differentiation: The analysis was done in the same manner as stated above for osteogenic differentiation except the use of adipogenic differentiation medium and the staining with Oil Red O for 15 minutes instead.
Chondrogenic differentiation: The analysis was done in the same manner as stated above for osteogenic differentiation except the use of cartilage differentiation medium and the staining with Toluidine blue for 30 min instead.

Observation Of Clone Formation Rate
Digest the P8 hUC-MSCs into single cells with 0.125% TrypLE, and collect the cells by centrifugation. After washing twice with saline and cell counting, plated 600 cells in three 10 cm cell culture dishes with 8 ml of medium per dish. The medium was change every 3 days. After culturing for 14 days, the cells were xed for 30 minutes, stained with gentian violet, and colony formation was observed under a microscope.

Cell Morphology Observation
Observe and record the cell expansion and cell morphology of different experimental groups every day under an inverted phase-contrast microscope.

Statistical analysis
All data was analyzed and processed with SPSS17.0 statistical software. The results were expressed as Mean ± SD. The comparison among multiple groups was done with one-factor analysis of variance, and the comparison between two groups was done with Student t-test.

Results
In this study, Wharton's jelly was isolated from selected freshly collected umbilical cord, shredded and adhered to culture asks. MSCBM, α-MEM and IMDM medium containing 5% platelet lysate were used for culture respectively. The medium was changed every 5 days. Up to 8 passage expansion were carried out.
The morphology, growth cycle, proliferation function, doubling time, differentiation function, and cell surface markers of different passages were studied.

Cell Morphology Observation And Proliferation Effects
First, cell morphology observation revealed that there were no signi cant differences in the morphology of P0 to P5 passage cells cultured in all three different basal media (P > 0.05). The difference in morphology was more signi cant after P5, showing that the MSCBM and α-MEM cultured cells had uniform morphology and sizes, radial, and obvious three-dimensional looking, On the other hand,IMDM cultured cells had larger areas than MSCBM andα-MEM cultured cells and the cells are at and uneven in shapes had rather poor three-dimensional looking,. The measurement results under microscope show that, from P6 to P8 passage, the volumes of IMDM cultured cells are larger than that of MSCBM and α-MEM cultured cells (0.01 < P < 0.05), while the MSCBM and α-MEM cultured cells showed no signi cant difference when compared with P0 to P5 cultures (P > 0.05) (Fig. 1).
By observing the same passage cell proliferation in different culture systems (Table 1), it was found that the numbers of cells obtained by MSCBM and α-MEM culture were higher than that of the IMDM (0.01 < P < 0.05), but there was no signi cant different between the rst two kinds of media (P > 0.05).The doubling time of P3, P5, and P8 passage cells (Table 2) indicated that it was signi cantly longer in IMDM than in MSCBM and α-MEM (0.01 < P < 0.05). There was no signi cant difference in cell doubling time between the latter two (P > 0.05).The combination of growth curve determination and cell doubling time calculation revealed that the cell proliferation functions of the three culture media are ranked as MSCBM, α-MEM, and IMDM from high to low. The cell proliferation function of MSCBM culture was signi cantly higher than that of IMDM (0.01 < P < 0.05), but was not signi cantly different from α-MEM (P > 0.05).
The clonal formation rate and the size of the formed clonal cell mass also re ect the cell proliferation functions ( Table 3). The number of clones formed by P8 cells cultured in different media are different (the cell cluster with more than 15 cells was counted as one cloned cell cluster). After 60 hours in culture, the clonal cluster numbers formed by the cells in different media, and the cell numbers in each clonal cluster are different. The clonal cluster numbers and the number of cells in each clone cluster formed by MSCBM cultured cells are higher than those formed by cells cultured in either α-MEM or IMDM (0.01 < P < 0.05).   . However, the cell numbers of adipocyte differentiation by MSCBM and α-MEM cultured P8 cells were signi cantly higher than that of the same-passage cells cultured with IMDM (0.01 < P < 0.05) (Fig. 2).

Comparison of cell surface marker detection
The detection of cell surface markers by ow-cytometry showed that cells of different passages cultured in IMDM, MSCBM and α-MEM all positively expressed CD73, CD90 and CD105 at ratios over 99%.

Discussion
MSCs are more and more used in clinical application and research,and has become a research hotspot. Therefore, there is a great demand for large-scale MSCs cultures. The cell culture methods used in various laboratories have also changed greatly. Many researchers have conducted experimental studies on the expansion of MSCs by changing the culture conditions and medium components. For example, the functions of MSCs under hypoxic culture conditions have been studied [17]. Simulated microgravity (SMG) can promote the differentiation of MSCs [18]. Some researchers have expanded MSCs by Simulate the microenvironment of stem cells in vivo [19]. These methods have resulted in higher quantity, high quality and high activity MSCs. MSC complete culture medium is composed of basal medium plus supplements. FBS is recognized as providing some essential cell growth nutrients and was the most commonly used MSC culture supplement. However, FBS is derived from cattle, thus containing xenogeneic animal proteins. There are also risks of infectious viruses such as mad cow disease, and other potential animal-derived diseases. The nutrient compositions of the serum-free culture systems are balanced and controllable, which can avoid some hidden dangers caused by xenogeneic animal-derived proteins, so it has been increasingly recognized and applied by researchers. However, it has not been successful in culturing MSC in serum-free medium with completely known components. Nowadays, the most commonly used MSC culture supplement is platelet lysate. Some companies provide platelet lysate products with added growth factors as MSC culture supplements. This type of culture systems is superior to using FBS in many ways as discussed. Therefore, it has been widely accepted. Cells cultured in this way also do not cause immunogenic reactions induced by xenogeneic sera to the body in the treatment of human diseases.
On the other hand, there are many types of basal media for MSCs growth, and they differ in components and content amounts. Different basal media therefore have very different effects on cell growth, proliferation and differentiation potential. This issue has not seemed to get enough research. In a recent study, αMEM and DMEM as basal media supplemented with 10% of human platelet lysate (hPL) and DMEM supplemented with 20% fetal bovine serum (FBS) and bFGF were compared in culturing adipose tissue-derived mesenchymal stromal cells (ASCs). The results indicate thatαMEM supplemented with 10% hPL yield the highest proliferation rate and rate of clonal genic potential, while no differences in adipocyte, osteocyte, and chondroblast differentiation [20]. This study was done with ASCs. As reported in the literature and in our own study, MSCs from different sources respond differently to cell culture basal media In this study, the three commonly used low-cost basal media: MSCBM, α-MEM, and IMDM were pared with commercial platelet lysate product UltraGROGAdvanced. It is designed to select a costeffective and low-cost basal medium to use in serum-free culture system for large-scale production of human umbilical cord MSCs.
Our results of comprehensive analysis of cell proliferation indicators include the number of cell expansions in the same culture cycle, cell doubling time, growth curve measurement and the clonal analysis, showing that the cell proliferation functions of the three basal media are arranged from high to low as MSCBM, α-MEM, IMDM. Among them, many indexes of MSCBM and α-MEM are very close, while that of IMDM is obviously worse than the other two media. The cells cultured in the three media all have strong differentiation functions, and can all differentiate into osteoblasts, adipocytes, and chondrocytes. It is worth noting that the number of MSCBM-cultured cells differentiated into adipocytes was higher than that of IMDM-cultured cells (0.01 < P < 0.05), but there was no signi cant difference from α-MEM-cultured cells. The cells cultured in the three media were equally highly expressed MSCs speci c markers CD73, CD90, CD105, and negatively expressed CD34, CD45. The percentage differences in the expression levels were not signi cant.
All three kinds of basal media are widely used. Although MSCBM is mainly distributed in China, its manufacture is actively expanding this product in other major markets. Based on the limited scope of the study,we also did not try to analyze the effects of individual components in the basal media.

Conclusion
In summary, based on our comprehensive analysis of cell proliferation indicators, also considering the cell morphology observation and differentiation potential analysis, we suggest the use of MSCBM or α-MEM supplemented with 5% UltraGROGAdvanced over the use of IMDM for cultivation of hUC-MSCs, especially for large-scale clinical applications. Our results should offer a good guidance on the selection of basal media for MSC cultures. The morphological observations and growth curves.