Isolation and Expansion of Mesenchymal Stem/Stromal Cells, Functional Assays and Long-Term Culture Associated Alterations of Cellular Properties

Mesenchymal stem cell/stromal cells (MSCs) can differentiate into a variety of cell types, including osteocytes, adipocytes and chondrocytes. MSCs are present in the multiple types of adult tissue, such as bone marrow, adipose tissue, and various neonatal birth-associated tissues. Given their self-renewal and differentiation potential, immunomodulatory and paracrine properties, and lacking major histocompatibility complex (MHC) class II molecules, MSCs have attracted much attention for stem cell-based translational medicine research. Due to a very low frequency in different types of tissue, MSCs can be isolated and expanded in vitro to derive sufficient cell numbers prior to the clinical applications. In this chapter, the methodology to obtain primary bone marrow-derived MSCs as well as their in vitro culture expansion will be described. To assess the functional properties, differentiation assays, including osteogenesis, chondrogenesis and adipogenesis, 3-D culture of MSCs and co-culture of MSCs and tumor cells are also provided. Finally, the long-term culture associated alterations of MSCs, such as replicative senescence and spontaneous transformation, will be discussed for better understanding of the use of MSCs at the early stages for safe and effective cell-based therapy.


Introduction
Mesenchymal stem/stromal cells (MSCs), a multipotent stem/progenitor cell type, were initially described in bone marrow by Friedenstein et al. as rapid adherence to tissue culture vessels and the discrete "fibroblast" colonies approximately 50 years ago [1,2]. Julius Cohnheim, a German-Jewish pathologist, firstly proposed that a fibroblast-like cell population for nonhematopoietic cells in bone marrow were involved in wound repair over 150 years ago [3]. In the late 1980s, Caplan firstly coined the name "mesenchymal stem cell (MSC)" [4]. Since then, MSCs have gained much attention over the last three decades. Many laboratories focusing on MSCs have developed diverse methods to isolate and expand MSCs from a variety of tissues. While the assessment of characteristics of MSCs is necessitated in different platforms/laboratories, most researchers come to acknowledge the lack of a universally accepted criteria to define MSCs. To address this question of cell equivalence, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) proposes three minimal criteria to define MSCs [5]: property of MSC plastic adherence, the expression of specific cellular surface antigen, and capacity for trilineage mesenchymal differentiation (osteogenesis, chondrogenesis and adipogenesis).
Human MSCs from different tissues have the varied phenotypic features, the morphologic inconsistency, and heterogeneous functional behavior [6][7][8]. Indeed, the properties of stem cell have not been well established yet. Due to the unknown in vivo multipotent properties of MSCs, the issue of MSC nomenclature remains actively controversial. In 2019, ISCT MSC committee issued a position statement on nomenclature of MSCs clarifying the functional definition to emphasize the functional distinction of mesenchymal stem versus stromal cells [9].
MSCs have been considered as a promising therapeutic tool in tissue engineering and regenerative medicine. MSCs are well known to be present in almost every type of adult tissues, such as bone marrow [10][11][12], adipose tissue [10,13,14], lung [11,15], synovial tissue [16,17], dental pulp and periodontal ligament [18]. Notably, it has become apparent that MSCs are identified in the various human embryonic tissues, such as fetal bone marrow [19], fetal liver [20], aorta gonad-mesonephros and yolk sac [21], as well as multiple neonatal birth-associated tissues, such as placenta [10,22,23], amniotic and chorionic membrane [23,24], umbilical cord tissue [10,[23][24][25], and umbilical cord blood [26,27]. Therefore, different platforms/laboratories may use different type of tissue sources and methodologies for isolation and expansion of MSCs. This chapter firstly outlines protocols for standardized isolation and expansion of human bone marrow-derived MSCs (BM-MSCs), a major source of human MSCs, as well as BM-MSCs' characteristics, cryopreservation and thawing. Protocols for the preparation of MSCs derived from the other tissue types are similar to that of BM-MSCs, except tissue sample processing differentially. Human BM-MSCs are estimated at a very low frequency at approximately 0.001-0.01% of total nucleated cells [28,29], and, therefore, human BM-MSCs are likely to be kind of difficult to isolate and harvest. This chapter will then focuses on optimal functional assays and application on the basis of our previous studies, which would be useful for researchers working with MSCs in basic research and translational and clinical applications, such as osteogenesis, chondrogenesis, adipogenesis, colony forming unit-fibroblast (CFU-F) assay, 3-D cellular co-culture, MSC homing and migration. Last but not least, the long-term culture associated alterations of MSCs' properties will be also discussed in this chapter. 11. Seed cells into one T-75 flask finally for 10 mL size marrow (a final cell concentration of 0.5-1.5 × 10 6 cells/cm 2 ). 12 mL of MSC growth medium is supplemented.
12. Put the flask in incubator at 37°C with 5% humidified CO 2 for 48 hours to allow cells to attach.
13. After 48 hours, observe with phase contrast microscopy and then remove growth medium and non-adherent cells.
14. Wash cells twice with pre-warmed medium and add 13 mL of fresh MSC growth medium. Return the flask to the incubator. 15. Change growth medium every 3 days and observe the cellular colony forming.
16. CFU-F become in the next 3-5 days. Continue to culture until the cells reach 80% confluence in the 2 weeks.
17. Remove the medium and wash with PBS 2-3 times.  32. Centrifuge at 400 × g for 5 minutes at room temperature. 33. Remove the supernatant without disturbing the cell pellet and resuspend the cells with an appropriate volume of pre-warmed PBS.
34. Wash and centrifuge at 400 × g for 5 minutes again. 35. Harvest cells. This culture is passage 1. MSCs at passage 1 can be frozen in liquid nitrogen (see the next) or continue to serially passage.
1. Remove the growth medium and wash cells with pre-warmed PBS twice.
2. Add the pre-warmed trypsin-EDTA solution to the flask. Return the flask to the incubator for 5 minutes.
9. Add 600 μL of lysis buffer (0.5% Triton-X 100 in molecular grade ddH 2 O) to each well and then scrape the cells off the surface using the end of a pipette tip. 13. Vortex samples and add 100 μL of each lysate to 15 mL tubes within 30 seconds. Fifteen minutes after the first sample is added, add 1 mL of 1 N NaOH to each tube in 30 second intervals removing tubes from water bath. The reaction will take place for 15 minutes at 37°C for each tube.
15. Add 300 μL of standards and samples in triplicate to a 96-well plate.
16. Measure the absorbance using excitation filter of 405 nm. One enzyme unit of ALP is defined as the quantity of enzyme which produces 1 nmol p-nitrophenol per 15 minutes [29].

Colorimetric quantitative calcium assay
1. Aspirate or pipette out all culture medium from each well of the 6-well culture plate that contains induced or control cells to be tested.  3. Add 125 μL of 0.5 N HCl to each well. 4. Scrape the cells off of the surface using a cell scraper and transfer the cells and HCl to a 1.5 mL polypropylene microcentrifuge tube with a tight fitting cap.
5. Add an additional 125 μL of 0.5 N HCl to each cell to recover any cells remaining in the well, and transfer this to the appropriate tube.
6. Samples may be capped tightly and stored at −20°C if they are not to be tested immediately.
7. Extract the calcium from the cells by shaking the tubes on an orbital shaker for 4 hours at 4°C. If using frozen samples, allow extra time for samples to thaw.
8. Centrifuge the sample tubes at 500 g for 2 minutes.
9. Carefully collect the supernatant with extracted calcium, without disrupting the cell pellets, and transfer these to a new tube. 11. Three μL of sample vs. 297 μL of assay reagent (1:100 ratio for sample to reagent) for 96-well plate is used for each calcium determination. Assay reagent is mixed by equal volume of two solutions (Color Reagent and Base Reagent) provided in the kit. Distribute the assay reagent by multipipettor after adding samples. Absorbance is read at 550 nm.
12. Unused sample extract may be re-frozen for future re-assay. If the reading was out of range, the sample can be diluted with ddH 2 O in a total volume of 3 μL (e.g. 1 μL of sample+2 μL of ddH 2 O) and re-assayed again.

3-D culture of human BM-MSCs and calcium deposition measured by von Kossa staining
PuraMatrix™ hydrogel (BD Biosciences) is a 16-amino acid synthetic peptide hydrogel composed of a repeating sequence of arginine, alanine, aspartate, and alanine (RADA16) [31], which is widely used for 3-D culture.
2. The stock of 1% peptide solution can be sonicated for 30 minutes to decrease its viscosity and then diluted with sterile ddH 2 O to a final concentration of 0.25% (w/v).
3. 300 μL of 0.25% gel solution is loaded into each well of the 24-well plate until it is uniformly spread.
4. The gelation is initiated by slowly dripping the medium along the wall of the well. 300 μL of medium is again added carefully on top and the plate is incubated at room temperature for one hour equilibration.

5.
After the peptide has assembled into hydrogel, the medium is changed two times over one hour to equilibrate the growth environment to physiological pH.
6. The equilibrated samples are stored overnight at 37°C incubator and the cells are seeded the next day.
7. Prepare the total of 4 × 10 4 cells suspended in MSC growth medium and 4 × 10 4 cells are seeded onto the hydrogel. The following day (Day 0), the medium will be replaced by osteogenic medium.
8. Von Kossa staining can be conducted at day 24 after differentiation [30]. MSCs are rinsed with the Tyrode's balanced salt solution and fixed with 10% buffered formalin (Fisher Scientific) for 30 minutes.
9. Incubated with 2% silver nitrate solution for 10 minutes in the dark.
10. Rinse with ddH 2 O and expose to light for 15 minutes.
11. Bright-field images of stained samples are captured with an inverted microscope.

3-D cellular culture conducted by encapsulation of human MSCs in PuraMatrix™ hydrogel
1. To generate a 0.25% final concentration of PuraMatrix™ hydrogel for cells encapsulation, one part of 1% PuraMatrix™ hydrogel is diluted with same volume of sterile 20% sucrose, to reach 0.5% PuraMatrix™ hydrogel in 10% sucrose, and then mix with one part of 2× concentration of cells resuspended in 10% sucrose.
2. For 24-well plates, 300 μL of PuraMatrix mixture is loaded into each well and 300 μL of medium is layered on top of the gel. The gelation of the PuraMatrix is completed in an incubator for 60 minutes.
3. Change medium the next day.
4. Von Kossa staining can also be conducted, as described above, or collect cells as follows for other experiments.
5. Mechanically disrupt BD PureMatrix™ and cells in the well or cell culture insert by pipetting the media and gel up and down.
6. Transfer to a 15 mL conical tube.
7. Rinse out the well or cell culture insert using PBS.
8. Centrifuge at 150 × g for 5 minutes. Discard supernatant. The pellet at the bottom of the tube contains cells and BD PuraMatrix fragments. 9. Resuspend pellet in 2 mL of PBS. Spin and collect pellet again.
10. Resuspend pellet in 1 mL of trypsin-EDTA and incubate at 37°C for 5-10 minutes. This will help separate cells that are still attached to each other.
11. Add 5 mL PBS to spin cell pellet again.
12. Aspirate the supernatant (do not disturb the gel). Resuspend pellet again in 1 mL of trypsin-EDTA and incubate at 37°C for 5-10 minutes.
13. Add 5 mL PBS to spin cell pellet again.
14. Aspirate the supernatant. Carefully take out one third of the gel pellet. Do not disturb the bottom (two-third) part of the gel.
15. Wash with PBS twice. 16. Add appropriate amount of lysis buffer to perform cell lysis and collect cell sample.

3-D cell pellet culture and chondrogenesis of human BM-MSCs
Chondrogenic   2. Position specimen in Peel-Away mold with 100% paraffin.
4. Specimen may be sectioned the following day.
Passage 4 human BM-MSCs are used for adipogenic differentiation. Adipogenic differentiation is induced by adipogenic medium.
1. Harvest cells from passage 4, as described in the previous section.

Resuspend cells in adipogenic medium carefully.
3. Transfer the single cell suspension in triplicate to 6-well plates (1 × 10 5 cells/well). 4. Culture cells in the incubator at 37°C with 5% humidified CO 2 for 3 weeks. 11. Observe and check the stained cells with phase contrast microscopy.

CFU-F assay
CFU-F assay can be used in vitro to evaluate the proliferation potential of MSCs. CFU-F assay is a well-established method for the quality control of MSCs' preparation. This section describes a traditional assay for CFU-F to evaluate the colony forming ability of human MSCs.

Collect cells from passage 4.
2. Prepare the single cell suspension in growth medium and seed cells in the 6-well plates in triplicate at three different densities, 1.5 × 10 5 , 2.5 × 10 5 , and 5 × 10 5 cells/well, in 2 mL growth medium, respectively.
3. Culture cells in the incubator at 37°C with 5% humidified CO 2 for two weeks.
4. Change medium twice each week and check with phase contrast microscopy daily to prevent overgrowth. Stop cell culture as soon as colonies are forming visibly and proceed with the Giemsa staining of CFU-F colonies on a benchtop as follows.
5. Wash the culture dishes twice with PBS.
6. Fix cells by adding 2 mL methanol to each well for 5 minutes at room temperature.
7. Gently remove the methanol and discard into the bio-hazardous waste.
8. Air dry the culture vessels and add the diluted Giemsa staining solution for 5-10 minute at room temperature.
9. Remove Giemsa staining solution and wash twice with ddH 2 O.
10. Count visible colonies manually with a diameter greater than 5 mm.

Co-culture of MSCs with cancer cell line
There are various 2-D or 3-D, dyeing or not dyeing, co-culture models of human MSCs and other cell sources to study cell-cell interaction, cell proliferation, MSCs' immunomodulatory capacity, and the cellular contribution of each cell type. These methods making co-cultures of MSCs and other cells are well-established, such as co-cultures of MSCs and human peripheral blood mononuclear cells [33,34], MSCs and T cells [35], MSCs and human hematopoietic stem cells [36], MSCs and umbilical vein endothelial cells [37]. MSC-cancer cell (PC-9) co-culture will be described in this section (Figure 3).
7. Wash with MSC growth medium once.
8. Add MSC growth medium and return the tissue culture vessel to the incubator.

MSC-PC-9 cell co-culture
1. Harvest MSCs with CellTrcker™ red dye and wash with PBS once.
2. Prepare the single cell suspension in growth medium at an appropriate cell concentration and transfer the cell suspension in a 6-well plate or a special chamber.
3. Culture cells in the incubator at 37°C with 5% humidified CO 2 for about 3 hours to allow cells to attach.
4. Change fresh growth medium slowly.
5. Add PC-9 cells in the 6-well plate (the same cell number of MSCs). Gently tap the side of the flask.
6. Return the 6-well plate to the incubator and culture cell overnight.
7. Check the cell co-culture under the contrast microscope and image under microscopy.

In vitro migration assay
The intercellular communication can be executed through a direct cell-cell interaction or through paracrine signaling mediated by a combination of active molecules. The major signaling molecules include cytokines, growth factors, chemokines, which can be generated and expressed in a wide variety of cell types including tumor cells and MSCs in response to multiple signals such as inflammatory or tumor microenvironment. Circulating MSCs are driven by such signaling molecules to home and subsequently migrate into the sites of tissue injury or disease. It is critical for the ability of MSCs to migrate and identify the injury sites for tissue repair and regeneration. Clinical data are still lacking for MSCs' homing and distribution of transplanted MSCs in the body, albeit a large number of in vivo studies are conducted on homing and migration pathways of MSCs for targeted stem cell-based therapies.
There are different approaches for improvement of MSC homing and migration. In this section, in vitro migration capacity of human BM-MSCs is evaluated by using an 8 μm-pore transwell chamber inserts (Corning).
1. Harvest MSCs and prepare cell suspension in the serum-free medium.
2. Transfer the cell suspension to the upper layer of a transwell insert at a density of 4 × 10 4 cells/cm 2 and allow cells to migrate to the lower compartment containing MSC growth medium overnight in the incubator at 37°C with 5% humidifies CO 2 .
3. Cells from the upper chamber of transwell are migrated. Gently scrape the MSCs using the cotton swab at the upper layer of the membrane.
4. The migrated MSCs at the lower layer are stained with 0.1% crystal violet.

Check the cells and image under a light microscope.
6. Count the number of stained MSCs manually.

Morphological and immunophenotypic alterations of MSCs
It is well known that MSCs demonstrate biological alterations in the course of in vitro long-term culture. Different tissue derived MSCs may present different morphological and immunophenotypic characteristics in the expansion culture. At present, there is lack of a unifying definition for the "passage" of MSCs. Morphological changes are continuous during the long-term culture and expansion of BM-MSCs [38][39][40], which display a fibroblast-like appearance at early passages while the flattened and larger morphology as well as a visible increase of cellular granularity in late passages. For example, one previous study showed that human BM-MSCs were consistent with a morphological appearance from passage 1 to passage 6-8 and beyond that period such cells became large and flat [40].
During further cultivation, MSCs demonstrate the altered common immunological surface markers. Comparison of the early and late passages of BM-MSCs reveals that no differences are observed between passage 2 and 6 MSCs in expression of CD44, CD90, CD105, HLA-ABC, and HLA-DR, while CD106 is downregulated in MSCs of passage 6 [41]. Research has also reported that the expression pattern of the common surface markers maintains consistently with consecutive passaging up to passage 8 of BM-MSCs [40]. In contrast, the positive expression of the common surface markers such as CD73, CD90 and CD105 presents at the passage 30 of human adipose-derived MSCs (AD-MSCs) [42] and human umbilical cord MSCs (UC-MSCs) [43].

Alterations of proliferation and differentiation of MSCs
MSCs exhibit a high proliferation rare at lower passage and, however, the rapid growth kinetics decrease gradually with consecutive cell passaging. A linear correlation is observed between cumulative population doubling and days in culture up to passage 6-8 of human BM-MSCs and the passage-dependent decrease in the proliferation rate is also observed beyond that period [40]. A reduction in the proliferation in the course of long-term cultivation has been reported in human dental pulp tissue-derived MSCs [44] and human tonsil-derived MSCs [45].
The differentiation ability of human BM-MSCs vary in long-term culture manifested by the significant reduction in expression levels of the osteogenic markers, such as ALP and osteocalcin, and adipogenic markers, such as fatty acid binding protein-4 and lipoprotein lipase at the late passages [45]. It has been reported that 25% samples of BM-MSCs from different donors in the 8th passage and the 20% in the 10th passage lost their osteogenic differentiation potential [46]. Similarly, 10% BM-MSC samples in the 6th passage, the 50% in the 8th passage, and the 60% in the 10th passage also lost their adipogenic differentiation [46]. In contrast, an in vitro differentiation study has also reported that the potential of adipogenesis decreases in higher passages (from the 5th passage) whereas the propensity for osteogenesis increases in the long-term culture [39].

Replicative senescence during long-term culture expansion of MSCs
Replicative senescence is known as the irreversible growth arrest of the mitotic cells and is induced by telomere shortening. The expression of senescence markers such as senescence-associated β-galactosidase, heterochromatin protein-1, and p16INK4a increase during aging [47,48]. Molecular damage and epigenetic alterations occur in aging stem cells [49], which can result in the impairment of stem cell function.
There are various signaling pathways involved in the senescence of MSCs, including oxidative damage [50,51], age-related defects [52], and senescence associated up-regulation of microRNAs [53]. MSC senescence can be observed with long-term in vitro cultivation [54,55], thus suggesting that a certain proportion of MSCs may undergo senescence during culture expansion. In vitro long-term culture of MSCs can induce continuous changes in gene expression [39,56]. The expression levels of the senescence related genes, such as p16, p21 and p53, increase gradually in MSCs in the course of in vitro culture expansion [57]. DNA-methylation changes in MSCs during long-term culture have been investigated as an important epigenomic feature of replicative senescence of MSCs [58][59][60]. DNA-methylation changes may affect the proliferation and differentiation of MSCs. Differential methylation patterns of gene and miRNAs show between early-passage (passage 5) and late-passage (passage 15) MSCs [60]. Some genes that are hypermethylated at passage 5 present the lower mRNA expression than does these hypermethylated at passage 15 and vise versa [60].
Senescent cells secrete a complex combination of interleukins, chemokines, growth factors, proinflammatory/inflammatory cytokines, which compose the senescence-associated secretory phenotype (SASP) [61,62]. One previous report has shown that conditioned medium (CM) collected from senescent BM-MSC culture at passage 10 is able to trigger senescence in young cells [63]. The key factors of senescent MSC CM needed for triggering senescence in the young MSCs have been characterized as insulin-like growth factor binding proteins 4 and 7, which are linked to cellular senescence and apoptosis [63]. Similarly, monocyte chemoattractant proten-1 (MCP-1), as a dominant component of the SASP, is markedly increased in the conditioned medium of the late-phase MSCs and MCP-1 treatment significantly increase the senescence phenotypes of umbilical cord blood-derived MSCs via its cognate receptor chemokine receptor 2 signaling cascade [64]. Senescence-associated changes are observed in the metabolome of MSCs during replicative senescence, including down-regulation of nicotinamide ribonucleotide and up-regulation of orotic acid, which may be used to monitor the cellular senescent state during culture expansion of MSCs [65].

In vitro long-term culture associated spontaneous transformation of MSCs
Sarcoma represents a very heterogeneous group of relatively rare tumors and a variety of different studies have investigated to support the MSC origin of sarcoma. There are a number of cellular and molecular mechanisms of MSC transformation for better understanding of MSCs' contribution to sarcomagenesis [66]. The majority of published research articles indicate that various sarcoma types have been shown MSCs' origin. Several group have reported spontaneous transformation in human and murine MSCs after long term culture [67][68][69][70]. For example, one study has reported that murine BM-MSCs are spontaneously transformed at passage 29 under standard conditions and that these transformed MSCs are able to generate fibrosarcoma in immunocompromised mice [70]. Accumulated chromosomal abnormalities, such as chromosome instability, chromosomal imbalances and aneuploidy, are suggested to be associated with the transformation of BM MSCs [70]. Indeed, chromosomal aberrations (chromosomal level) in in vitro cultures of human MSCs have been reported in previous studies, including human BM-MSCs after passage 4 [71], human AD-MSCs from passage 5 [72], and UC-MSCs from passage 5 [73].
There are also studies that have not detected the transformation of MSCs in long-term culture [74][75][76]. One previous study reports that human BM-MSCs do not undergo malignant transformation after long-term in vitro culture for up to 44 weeks and these cells maintain a normal karyotype [75]. In agree with the previous report [75], another study has described the occurrence of aneuploidy in cultivated human MSCs without evidence of transformation either in vitro or in vivo [76]. In addition, Røsland G. V. et al have reported that human BM-MSC spontaneous transformation phenomenon occurred in consequence of the cross-contamination between the transformed human MSCs and human cancer cells [77]. To date, there is no solid evidence for the transformation of different sarcoma subtypes from MSCs and it leaves an uncertainty for MSCs with the ability to spontaneously transform.

Conclusion and perspective
MSCs provide huge opportunities in translational medicine for treatment of a range of diseases or medical conditions. MSCs are multipotent stem cells and such cells can be isolated from various tissues including bone marrow, a major source of human MSCs. Given that a large number of MSCs are required for the clinical application, in vitro expansion of MSCs is critical. However, MSCs at higher passage could lead to the culture-associated alterations, such as cellular morphology, immunological surface markers, proliferation, differentiation, and cell genetics. Due to in vitro long-term culture associated spontaneous transformation of MSCs, the safety of MSC therapy remains the major concerns. Human MSCs from various tissues present the varies biological properties. At present, consensus is lacking regarding materials and culture protocols, culture conditions, supplement of growth factors,

Conflict of interest
No competing interests for this work.