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Three dimensional microcarrier system in mesenchymal stem cell culture: a systematic review

Cell & Bioscience volume 10, Article number: 75 (2020) Cite this article

Abstract

Stem cell-based regenerative medicine is a promising approach for tissue reconstruction. However, a large number of cells are needed in a typical clinical study, where conventional monolayer cultures might pose a limitation for scale-up. The purpose of this review was to systematically assess the application of microcarriers in Mesenchymal Stem Cell cultures. A comprehensive search was conducted in Medline via Ebscohost, Pubmed, and Scopus, and relevant studies published between 2015 and 2019 were selected. The literature search identified 53 related studies, but only 14 articles met the inclusion criteria. These include 7 utilised commercially available microcarriers, while the rest were formulated based on different surface characteristics, all of which are discussed in this review. Current applications of microcarriers were focused on MSC expansion and induction of MSCs into different lineages. These studies demonstrated that MSCs could proliferate in a microcarrier culture system in-fold compared to monolayer cultures, and the culture system could simulate a three-dimensional environment which induces cell differentiation. However, detailed studies are still required before this system were to be adapted into the scale of GMP manufacturing.

Introduction

Mesenchymal stem cells

Adult mesenchymal stem cells are becoming increasingly popular as a potential cell source in regenerative medicine nowadays. This multipotent CD 34− fibroblast-like stem cell has the ability to differentiate into specialized cells such as adipocytes, osteocytes, chondrocytes, and myocytes [1,2,3]. It can be isolated from various adult tissue sources such as blood or adipose tissue, dermis, muscle, dental pulp, and Wharton’s jelly [4,5,6,7]. In contrast to embryonic pluripotent stem cells, MSC is devoid of ethical, histocompatibility, and teratomas-formation issues. In addition to that, several studies successfully demonstrated the efficacy of MSCs in regenerating new tissues and repair defects [8,9,10,11].

Stem cell-based regenerative medicine is an emerging approach for tissue reconstruction. Allogenic hematopoietic stem cell transplant has the potential to play a significant role in the treatment of autoimmune diseases or hematopoietic disorders. However, the applications of therapy are limited due to morbidity and mortality of graft versus host disease (GVHD). Studies have reported that mesenchymal stem cells could reduce inflammatory cytokines through interplay with several subsets of immune cells; thus the immunoregulatory capacity of MSCs makes them of great interest in clinical studies involving GHVD [12,13,14].

Anti-inflammatory properties of mesenchymal stem cells

Aside from its regenerative capabilities, MSCs are known for its immunosuppression or anti-inflammatory ability in cell transplantations. The role of MSCs as an anti-inflammatory agent has become more evident with the elucidation of the mechanism of inflammation, which includes the release of intracellular cytokines such as interleukin-1α from injured cells or activation of macrophages by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) interaction with receptors to generate proinflammatory cytokines [15,16,17].

According to the results reported by [18], administration of MSCs into a mouse model successfully inhibited bleomycin (BLM)-induced elevation of TNF-α, IL-1α, and IL1RN mRNA in the lungs, which protected lung tissues from BLM-induced injury by blocking TNF-α and IL-1α, the main proinflammatory cytokines in the lungs. A similar anti-inflammatory property was reported by Oh et al., where the suppression of IL-2 and IFN-γ, and the reduced infiltration of CD4+ cells by MSCs, showed a reduction in corneal inflammation and neovascularisation [19]. In short, the anti-inflammatory effects of MSCs have been reported in various events such as lung injury, myocardial infarction, corneal injury, sepsis, and diabetic wound healing [20,21,22,23].

Cytokines in inflammatory events

Inflammatory mechanisms in GVHD were generally associated with activation of immune cells (T cells, B cells, and macrophages) in the presence of antigen-presenting cells (APC). These immune cells will release substances called cytokines which regulate or facilitate immune responses. For instance, the IL-1 pathway plays a crucial role in generating sterile inflammation, which is similar in effect as that produced by tumor necrosis factor-α (TNF-α) in lung injuries [24]. In addition, the presence of pro-inflammatory cytokines such as TNF-α and IL-6 in serum also contributed to sepsis in a mouse model [25]. In addition to that, the secretion of TNF-α and IL-1α by macrophages also induced peritonitis in a mouse model [26].

TNF-α is a prototypical member of a large superfamily known as TNF/TNFR superfamily, which comprises more than 40 family members. The TNF-α gene is a single-copy gene on human chromosome 6 (murine chromosome 17), which codes for a 27-kDa (233 amino acid) protein that is proteolytically cleaved into a 17-kDa (157 amino acid) molecule [27]. TNF-α is secreted from activated macrophages by induction of Toll-like receptors and other factors, and generally after priming with interferon gamma (IFN-γ). It is rapidly released after trauma, infection, or exposure to bacterial-LPS and was shown to be one of the early abundant mediators in inflamed tissues. Apart from that, the role of TNF-α during inflammation is mostly associated with coordination of the pro-inflammatory cytokine cascade. Therefore, TNF-α is considered as a master regulator of pro-inflammatory cytokines during inflammation [28].

Mesenchymal stem cells expansion

Clinical applications of mesenchymal stem cells require billions of cells [29] and two-dimensional platforms, which might pose a challenge in scaling-up. In a clinical study of acute ischemic stroke, it was suggested that the number of MSCs required for administration to a single patient ranged from 1–8 × 106 MSCs per kg of body mass, depending on the indication [30]. Innovation of cell culture products aim to address surface limitations imposed by monolayer culture flasks. Multi-layered flasks which could accommodate up to 40 layers of culture chambers is a good example of such innovation. However, difficulty in observing the in-cultured cells could be a potential downside of this innovation. In order to achieve a scalable undifferentiated mesenchymal stem cell number for cell transplantation and tissue engineering applications, 3-dimension culture techniques seem to be a more reliable approach compared to 2D cultures. Mesenchymal stem cell expansion in bioreactors potentially provide ease of scalability, flexible modes of operation, better process monitoring, and control compatibility. For example, Zhou et al. (2013) developed a novel strategy for 3D expansion of bone marrow MSCs, which produced a 10.4 ± 0.8-fold increase compared to 2D cultures on day 5.

3-D cell culture

Various tissue-engineering studies utilising a 3D scaffold system have shown their efficacy in in vitro culture of MSCs. Three-dimensional culture conditions simulates environment of cells in vivo, therefore providing a suitable condition that enhances cellular activities that are not observed in normal monolayer cultures [31].

While 3D scaffold systems propose unique attractive advantages, these also brought about significant challenges for MSC culture including: (i) the use of undefined components from human or animal tissue, which may result in batch-to-batch variation and poses risks for pathogen and immunogen transfer [32, 33], and thus an obstacle for good manufacturing practice (GMP) in cell production [34]; (ii) substantial cell aggregation that could possibly lead to MSC differentiation or senescence [35]; (iv) limited cell expansion rates and yield per volume [36]; and (v) unpredictable consequences of long-term serial expansion.

One way to address a few of the abovementioned challenges is to adapt the use of microcarriers. These micron-sized spherical particles were initially used for the growth of adherent cells for viruses and production of vaccines [37,38,39]. Over the decades, properties of microcarriers underwent various modification and innovation to meet the need of different cell types. To date, there are numerous manufacturers and multiple microcarrier varieties are commercially available.

Microcarrier in 3-D culture

Microcarriers provide surface matrices that enable attachment of adherent cells to form cell-microcarrier complexes suspended in growth medium [40]. The fundamental structure of microcarriers are tiny beads (size ranging from 100–300 microns) that are able to maintain suspension during stirring. A number of microcarriers have been synthesized and made commercially available, e.g. glass, diethylaminoethyl (DEAE)-dextran, acrylamide, polystyrene, collagen, and alginate [41].

Microcarrier-based cell culture systems are relatively flexible as they promote higher cell yield and can be integrated into existing bioprocess manufacturing systems such as stirred bioreactors and spinner flasks [42]. Such microcarriers have been established for vaccine production or fermentation processes decades ago, however, downstream processes were only focused on metabolites instead of cells. In cell-based therapy, the product of interest are the cells itself, and the main objective of bioprocessing changed from maximising the yield of metabolites to harvesting large quantities of MSCs. Since mesenchymal stem cells required a support surface for cell division, microcarriers are often added into culture media to provide sufficient adherent surface for MSCs in three-dimensional culture. Figure 1 shows the basic approach of up-scaling MSC production in microcarrier-based culture system. Microcarriers provide a large surface area for cell growth during proliferation in suspension cultures, thus allowing scaling-up of cell production in small volumes of medium [43]. In addition to that, the suspended system provides better nutrient intake and gas exchange, and at the same time the adjustable stirring mechanism provides control over shear stress which might facilitate differentiation along certain lineages [44]. This approach could be an ideal model for MSC expansion for its large surface area per unit volume of media compared to T-flask cultures. Hence, the selection of microcarriers are crucial as it would contribute a direct impact on cell expansion.

Fig. 1
figure 1

Schematic illustrating the basic flow of up-scaling MSC culture from laboratory scale into manufacturing scale. To date, a “pre-adaptation” period prior microcarrier culture system in MSC is still required, where 2-D culture flasks were used for cell isolation. The up-scale of MSC production can be first optimising culture condition in a small-scale culture system (usually 10–500 mL), followed by up-scaling into bench scale (1–5 L), and finally up to manufacturing scale (up to 20 L). There are 3 major types of microcarrier: non-porous, microporous and macroporous. Cells attach differently based on the porosity of the microcarrier. In general, cells will be attached on the surface of non-porous and microporous microcarrier; while microporous microcarrier provides larger spaces, which allow cells to attach into the inner part of the microcarrier. Due to the similarity towards human body environment, MSCs-microcarriers constructs were found to be able to differentiated into osteo- and chondro-lineage in a specific condition

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To date, there are vast reports which suggests extensive choices of suitable microcarriers for mesenchymal stem cell culture. Alginate/PEG-based microcarriers could provide good attachment and proliferation of human umbilical cord blood mesenchymal stem cells, with well-controlled microcarrier degradation for harvesting [45]. The use of Cytodex type 1 from GE healthcare for porcine bone marrow-derived MSCs could produce cell numbers of approximately 4 × 105 cells/mL [46], while the use of Cytodex type 3 showed similar cell numbers (3.8 × 105 cells/mL) for human placental MSCs [47].

Methods

Search strategy

The review was conducted to systematically assess articles on the application of microcarriers for MSC culture. Three databases were comprehensively used to search for relevant studies; Medline via Ebscohost, Pubmed, and Scopus. The keywords used were the combination of words “Mesenchymal Stem Cell” AND “Microcarriers”.

Selection criteria

The year limit for searches was from 2015 to 2018, and only studies published in English were considered. The search outcomes identified all articles containing the word “mesenchymal stem cell” and “microcarrier”. Databases were searched individually to ensure all relevant studies were considered. The titles and abstract were carefully screened for eligibility related to the topic of interest. Primary studies related to microcarrier application were included. Review articles, news articles, letters, editorials, and case studies were excluded from the search.

Data extraction and management

Data were extracted from each eligible article by two reviewers. The selected papers were screened in several phases prior to inclusion. First, titles that were not relevant to the topic were excluded. Next, abstracts of the papers were screened, and unrelated studies were excluded. All duplicates were removed. The following data were summarized from the selected studies: authors, year, source of MSCs, applications, type of microcarrier used, results, and conclusion.

Results

Search result

The primary search identified 432 articles: 61 articles were derived from Pubmed, 265 from Ebscohost, and 106 articles from Scopus. To minimize bias and improve the strength of the related articles, two reviewers independently assessed the articles according to the inclusion and exclusion criteria. A total of 379 articles were removed as they were unrelated to either mesenchymal stem cells or microcarriers. A joint discussion was conducted to achieve consensus on differences which emerged during the assessment. From the 53 remaining articles, 12 duplicates were removed before full articles were retrieved. From the remaining 41 articles, 27 articles were rejected based on the inclusion criteria as these articles were not primary studies, were not related to mesenchymal stem cells or microcarriers, or were unavailable as full articles. Finally, a total 14 studies were selected for data extraction in this review. The flow chart of the selection process is shown in Fig. 2.

Fig. 2
figure 2

Flow chart of the article selection process from Pubmed, Ebscohost, and Scopus databases

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Study characteristics

All studies were published between 2015 and 2019 and reported on in vitro studies. Thirteen studies utilised human mesenchymal stem cells, while only one reported using rat MSCs. Seven out of 14 articles utilised commercially available microcarriers, while the rest were formulated based on different surface characteristics. From the generated data, articles were classified into three aspects: Microcarriers in MSC culture, MSC expansion and MSC differentiation. A summary of the studies is provided in Table 1.

Table 1 Summary and classification of the 14 articles selected from the database search
Full size table

Discussion

The database search provided 14 articles related to Wharton’s Jelly and microcarrier. From these articles, various sources were examined regarding microcarrier application on MSC culture. This review assessed the application of microcarrier on MSC culture, which may have remarkable potential for different usage in future application.

Microcarrier in MSC culture

Generally, microcarriers can be divided into 3 major types: non-porous, microporous and macroporous (Fig. 1). While non-porous microcarriers are relatively straightforward with limited surface area, the microporous structure of microcarriers allow cells attached on the carrier to undergo material transfer on the basolateral side of the cell; however, the surface area available for cell attachment is also limited on the outer surface of the microcarrier. In contrast, macroporous microcarriers provide a larger pore size that enable cells to enter into the microcarrier. In this case, macroporous microcarriers contributes a larger surface area per millilitre of media compared to microporous microcarriers, hence potentially higher cell yields in large scale cultures [48]. Table 2 shows the summary of the microcarrier used in the 14 studies selected aforementioned, the details were arranged based on the material, surface feature, diameter, porosity, concentration in culture and results of application for each microcarriers.

Table 2 Summary and classification of the microcarrier used in the 14 studies selected
Full size table

The fabrication material of microcarriers is also a crucial factor in microcarrier cultures because of its physical and chemical effects towards cells, which include porosity, mechanical strength, permeability of nutrients, size, density, and shape [49]. In order to facilitate adherent cells to attach on the carrier surface, the divalent cations or protein available in culture medium is important so that cell could utilise it for attachment. Polymers such as polystyrene, plastic, or glass are commonly utilised as the basic matrix of microcarriers; these microcarriers are usually positively charged or chemically bounded to facilitate the attachment of adherent cells which possess an uneven distribution of negative surface charge. While microcarriers with higher charge densities were developed to promote cell adhesion for cell lines with weak adhesion (E.g. Cytopore 1 & 2), these microcarriers poses a challenge during cell harvesting due to difficulties with cell detachment at the end of the culture [50].

To overcome this problem, biopolymers (dextran, gelatin, cellulose, agarose, alginate) were introduced as they potentially facilitate cell harvesting while providing a biocompatible environment for cultures [51, 52]. In addition, microcarriers with surface modifications (E.g. protein or collagen coated), could also achieve a similar effect as the microcarriers mentioned above. Fibronectin for example, is commonly used to coat plastic or glass microcarriers to increase cell adhesion in microcarrier cultures, and used in concentrations ranging from 1–50 ug/mL [53,54,55]. On the other hand, compounds such as casein, chitosan, or even PNIPAAm was grafted on the surface of microcarriers to modify its adhesion properties and to provide an easier solution for cell harvesting [51, 56, 57].

Application of microcarriers in MSC culture

MSC expansion

A study found that PEG coated microcarriers supported the expansion of hMSCs in a serum-free environment, with doubling time under 25 h in the growth phase, as well as preserving its osteogenic and adipogenic differentiation post-harvest [58]. Genipin cross-linked alginate-chitosan microcarriers were demonstrated to provide 26% higher MSC attachment and twice the proliferation rate compared to the commercial microcarrier, Cytodex 1. The cells produced were easily detached without an extended incubation period or intense agitation during harvesting [51]. Whereas Krutty et al. developed a chemically defined PVG microcarrier which achieved a six-fold expansion in MSCs, while retaining their ability to differentiate after harvesting [59].

Under xenogenic-free culture conditions, Gupta et al. reported that HPL resulted in faster cell proliferation by 5.2 ± 0.61-fold in comparison to 2.7 ± 02.22-fold in FBS [60]. In addition, an automated serum-free, microcarrier culture system was established. It was found that such approach can produce more than tenfold MSC expansion compared to serum-based, manual spinner flask methods [61].

Several studies have been conducted on the formation of MSC-microcarrier aggregates and explored possible methods to overcome drawbacks associated with such culture strategies. It was suggested that hMSC aggregates generated from thermal responsive microcarriers in bioreactors maintained comparable immunomodulation and cytokine secretion compared to conventional culture strategies [62]. Heathman and co-workers reported a minimum agitation speed in a bioreactor system to obtain high cell numbers; however, low agitation were still accompanied by cell aggregation, leading to inconsistencies between pre- and post-harvest sampling. Therefore, an alternative oxygen supply method is needed to overcome the current downsides faced by readily available methods, which introduced shear forces to the cells during increased agitation speeds in up-scaling of cultures [63]. On the other hand, a protocol which utilised short periods of intense agitation in the presence of enzymes such that the cells were detached yet remained undamaged and retained post-harvest characteristics, was reported [54].

MSC differentiation

Aside from up-scaling MSC expansion, more researchers were shifting their focus towards inducing cell differentiation in microcarrier cultures simulating a three-dimensional human body environment. Lin et al. showed that chondrogenic pellets generated from microcarrier cultures developed larger pellet diameters, and produced more DNA, GAG and collagen II per pellet with greater GAG/DNA and collagen II/DNA ratios compared with that of tissue culture flasks, while similar result were observed by using another type of microcarrier [64]. An increasing number of studies have highlighted bone formation potential by using microcarrier cultures, for example, a new process developed by Zhang et al. fabricated pre-vascularized bone microtissues by integrating microcarrier culture and co-culture with MS and HUVEC [65]. Aside from that, Tanimowo et al. fabricated a novel agarose-k-casein microsphere which upregulated the expression of osteogenic differentiation markers in bone marrow MSCs [57]. A titanium phosphate glass microcarrier that enhances bone morphogenic protein-2 (BMP-2) and osteopontin (OPN) expression by h-MSC was introduced. BMP-2 is considered an important protein in cell differentiation and tissue regeneration, which is normally associated with osteoinductive growth factors [66]; OPN on the other hand is mainly related to bone metabolism and remodelling [67]. In this case, it was suggested that titanium phosphate glass microcarriers influenced hMSC differentiation and metabolic activity and could contribute in bone tissue engineering [55].

Conclusion

Limitation of cell numbers in MSC-based cell therapy enlightened multiple approaches to increase the cell yield. Three-dimensional microcarrier cultures seems to be a potential candidate in the up-scaling production of MSCs. This review demonstrates that microcarriers, whether commercially available or produced in-house, were capable of enhancing production and inducing chondrogenic and osteogenic differentiation in MSCs.

However, several challenges in this system need to be addressed during cell manufacturing. The yields of MSC up-scale activity are still showing inconsistency from one another, even similar culture techniques and consumables were used. This problem could be possibly due to the batch-to-batch variances present in undefined media which relying on animal/human derived serum as main supplement. The variation from each batches of serum further affect the quality of the up-scaled product by different sources of origin, brands, and present of unidentified risk of contamination. In this case, one of the solutions to minimise this variations is the adaptation of serum free media (SFM) in MSC culture as mentioned by Ota et al. [68]. Aside from cell yield variations, the downstream harvesting approaches still require optimisation to improve cell recovery; in fact, MSC differentiation efficiency in 3D system remains uncertain and the mechanism is still not well-studied. Therefore, detailed studies are still required before this system to be adopted into the scale of GMP manufacturing.

Availability of data and materials

The datasets generated during and/or analysed during the current study are available in the Scopus, Ebscohost, and Pubmed repository.

Abbreviations

MSC:

Mesenchymal stem cell

GVHD:

Graft versus host disease

PAMPs:

Pathogen-associated molecular patterns

DAMPs:

Damage-associated molecular patterns

BLM:

Bleomycin

TNF:

Tumor necrosis factor

IL:

Interleukin

IFN:

Interferon

APC:

Antigen-presenting cell

GMP:

Good manufacturing practice

DEAE:

Diethylaminoethyl

PEG:

Polyethylene glycol

HPL:

Human platelet lysate

GAG:

Glycosaminoglycan

References

  1. Abomaray FM, Al Jumah MA, Kalionis B, AlAskar AS, Al Harthy S, Jawdat D, et al. Human chorionic villous mesenchymal stem cells modify the functions of human dendritic cells, and induce an anti-inflammatory phenotype in CD1 + dendritic cells. Stem Cell Rev Reports. 2015;11(3):423–41.

    Article  CAS  Google Scholar 

  2. Chen X, Armstrong MA, Li G. Mesenchymal stem cells in immunoregulation. Immunol Cell Biol. 2006;84(5):413–21. https://doi.org/10.1111/j.1440-1711.2006.01458.x.

    Article  CAS  PubMed  Google Scholar 

  3. Keating A. Mesenchymal stromal cells. Curr Opin Hematol. 2006;13:419–25.

    Article  PubMed  Google Scholar 

  4. Maharlooei MK, Bagheri M, Solhjou Z, Jahromi BM, Akrami M, Rohani L, et al. Adipose tissue derived mesenchymal stem cell (AD-MSC) promotes skin wound healing in diabetic rats. Diabetes Res Clin Pract. 2011;93(2):228–34.

    Article  PubMed  Google Scholar 

  5. Baba K, Yamazaki Y, Ikemoto S, Aoyagi K, Takeda A, Uchinuma E. Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived autoserum. J Craniomaxillofac Surg. 2012;40(8):768–72.

    Article  PubMed  Google Scholar 

  6. O’Donoghue K. Identification of fetal mesenchymal stem cells in maternal blood: implications for non-invasive prenatal diagnosis. Mol Hum Reprod. 2003;9(8):497–502.

    Article  PubMed  Google Scholar 

  7. Kerkis I, Ambrosio CE, Kerkis A, Martins DS, Zucconi E, Fonseca SA, et al. Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: local or systemic? J Transl Med. 2008;6(1):35. https://doi.org/10.1186/1479-5876-6-35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Vojtaššák J, Danišovič L, Kubeš M, Bakoš D, Jarábek L, Uličná M, et al. Autologous biograft and mesenchymal stem cells in treatment of the diabetic foot. Neuroendocrinol Lett. 2006;27:134–7.

    PubMed  Google Scholar 

  9. Petite H, Viateau V, Bensaïd W, Meunier A, De Pollak C, Bourguignon M, et al. Tissue-engineered bone regeneration. Nat Biotechnol. 2000;18(9):959–63.

    Article  CAS  PubMed  Google Scholar 

  10. Takewaki M, Kajiya M, Takeda K, Sasaki S, Motoike S, Komatsu N, et al. MSC/ECM cellular complexes induce periodontal tissue regeneration. J Dent Res. 2017;96(9):984–91.

    Article  CAS  PubMed  Google Scholar 

  11. Ren L, Ma D, Liu B, Li J, Chen J, Yang D, et al. Preparation of three-dimensional vascularized MSC cell sheet constructs for tissue regeneration. Biomed Res Int. 2014;2014:301279.

    PubMed  PubMed Central  Google Scholar 

  12. Lim JY, Ryu DB, Lee SE, Park G, Min CK. Mesenchymal Stem Cells (MSCs) attenuate cutaneous sclerodermatous graft-versus-host disease (Scl-GVHD) through inhibition of immune cell infiltration in a mouse model. J Invest Dermatol. 2017;137(9):1895–904.

    Article  CAS  PubMed  Google Scholar 

  13. Wang L, Gu Z, Zhao X, Yang N, Wang F, Deng A, et al. Extracellular vesicles released from human umbilical cord-derived mesenchymal stromal cells prevent life-threatening acute graft-versus-host disease in a mouse model of allogeneic hematopoietic stem cell transplantation. Stem Cells Dev. 2016;25(24):1874–83. https://doi.org/10.1089/scd.2016.0107.

    Article  CAS  PubMed  Google Scholar 

  14. Berger M, Mareschi K, Castiglia S, Rustichelli D, Mandese A, Migliore E, et al. In vitro mesenchymal progenitor cell expansion is a predictor of transplant-related mortality and acute GvHD III-IV after bone marrow transplantation in univariate analysis: a large single-center experience. J Pediatr Hematol Oncol. 2019;41(1):42–6.

    Article  PubMed  Google Scholar 

  15. Prockop DJ, Youn OhJ. Mesenchymal stem/stromal cells (MSCs): role as guardians of inflammation. Mol Ther. 2012;20:14–20.

    Article  CAS  PubMed  Google Scholar 

  16. Soehnlein O, Lindbom L. Phagocyte partnership during the onset and resolution of inflammation. Nat Rev Immunol. 2010;10:427–39.

    Article  CAS  PubMed  Google Scholar 

  17. Rock KL, Latz E, Ontiveros F, Kono H. The Sterile Inflammatory Response. Annu Rev Immunol. 2010;28(1):321–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ortiz LA, DuTreil M, Fattman C, Pandey AC, Torres G, Go K, et al. Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury. Proc Natl Acad Sci USA. 2007;104(26):11002–7.

    Article  CAS  PubMed  Google Scholar 

  19. Oh JY, Kim MK, Shin MS, Lee HJ, Ko JH, Wee WR, et al. The anti-inflammatory and anti-angiogenic role of mesenchymal stem cells in corneal wound healing following chemical injury. Stem Cells. 2008;26(4):1047–55. https://doi.org/10.1634/stemcells.2007-0737.

    Article  CAS  PubMed  Google Scholar 

  20. Luo G, Cheng W, He W, Wang X, Tan J, Fitzgerald M, et al. Promotion of cutaneous wound healing by local application of mesenchymal stem cells derived from human umbilical cord blood. Wound Repair Regen. 2010;18(5):506–13. https://doi.org/10.1111/j.1524-475X.2010.00616.x.

    Article  PubMed  Google Scholar 

  21. Jeon YK, Jang YH, Yoo DR, Kim SN, Lee SK, Nam MJ. Mesenchymal stem cells’ interaction with skin: wound-healing effect on fibroblast cells and skin tissue. Wound Repair Regen. 2010;18(6):655–61. https://doi.org/10.1111/j.1524-475X.2010.00636.x.

    Article  PubMed  Google Scholar 

  22. Smith AN, Willis E, Chan VT, Muffley LA, Isik FF, Gibran NS, et al. Mesenchymal stem cells induce dermal fibroblast responses to injury. Exp Cell Res. 2010;316(1):48–54.

    Article  CAS  PubMed  Google Scholar 

  23. Rodriguez-Menocal L, Shareef S, Salgado M, Shabbir A, Van Badiavas E. Role of whole bone marrow, whole bone marrow cultured cells, and mesenchymal stem cells in chronic wound healing. Stem Cell Res Ther. 2015;6(1):1–11.

    Article  CAS  Google Scholar 

  24. Eigenbrod T, Park J-H, Harder J, Iwakura Y, Núñez G. Cutting edge: critical role for mesothelial cells in necrosis-induced inflammation through the recognition of IL-1α released from dying cells. J Immunol. 2008;181(12):8194–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Németh K, Leelahavanichkul A, Yuen PST, Mayer B, Parmelee A, Doi K, et al. Bone marrow stromal cells attenuate sepsis via prostaglandin E 2-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med. 2009;15(1):42–9.

    Article  CAS  PubMed  Google Scholar 

  26. Choi H, Lee RH, Bazhanov N, Oh JY, Prockop DJ. Anti-inflammatory protein TSG-6 secreted by activated MSCs attenuates zymosan-induced mouse peritonitis by decreasing TLR2/NF-κB signaling in resident macrophages. Blood. 2011;118(2):330–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Spriggs DR, Deutsch S, Kufe DW. Genomic structure, induction, and production of TNF-alpha. Immunol Ser. 1992;56:3–34.

    CAS  PubMed  Google Scholar 

  28. Maini RN, Elliott MJ, Brennan FM, Feldmann M. Beneficial effects of tumour necrosis factor-alpha (TNF-α) blockade in rheumatoid arthritis (RA). Clin Exp Immunol. 1995;101:207–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Rowley J, Abraham E, Campbell A, Brandwein H, Oh S. Meeting lot-size challenges of manufacturing adherent cells for therapy. Vol. 10, BioProcess International. 2012. www.corning.com/lifesciences/whatsnew. Accessed 2 Jan 2020.

  30. Díez-Tejedor E, Gutiérrez-Fernández M, Martínez-Sánchez P, Rodríguez-Frutos B, Ruiz-Ares G, Lara ML, et al. Reparative therapy for acute ischemic stroke with allogeneic mesenchymal stem cells from adipose tissue: a safety assessment: a phase II randomized, double-blind, placebo-controlled, single-center, pilot clinical trial. J Stroke Cerebrovasc Dis. 2014;23(10):2694–700.

    Article  PubMed  Google Scholar 

  31. Levenberg S, Burdick JA, Kraehenbuehl T, Langer R. Neurotrophin-induced differentiation of human embryonic stem cells on three-dimensional polymeric scaffolds. Tissue Eng. 2005;11(3–4):506–12.

    Article  CAS  PubMed  Google Scholar 

  32. Chen VC, Couture SM, Ye J, Lin Z, Hua G, Huang HIP, et al. Scalable GMP compliant suspension culture system for human ES cells. Stem Cell Res. 2012;8(3):388–402.

    Article  CAS  PubMed  Google Scholar 

  33. Zweigerdt R, Olmer R, Singh H, Haverich A, Martin U. Scalable expansion of human pluripotent stem cells in suspension culture. Nat Protoc. 2011;6(5):689–700.

    Article  CAS  PubMed  Google Scholar 

  34. Halme DG, Kessler DA. FDA regulation of stem-cell–based therapies. N Engl J Med. 2006;355(16):1730–5. https://doi.org/10.1056/NEJMhpr063086.

    Article  CAS  PubMed  Google Scholar 

  35. Chen AKL, Chen X, Choo ABH, Reuveny S, Oh SKW. Critical microcarrier properties affecting the expansion of undifferentiated human embryonic stem cells. Stem Cell Res. 2011;7(2):97–111.

    Article  CAS  PubMed  Google Scholar 

  36. Serra M, Correia C, Malpique R, Brito C, Jensen J, Bjorquist P, et al. Microencapsulation technology: a powerful tool for integrating expansion and cryopreservation of human embryonic stem cells. PLoS ONE. 2011;6(8):e23212. https://doi.org/10.1371/journal.pone.0023212.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Van Hemert P, Kilburn DG, Van Wezel AL. Homogeneous cultivation of animal cells for the production of virus and virus products. Biotechnol Bioeng. 1969;11(5):875–85. https://doi.org/10.1002/bit.260110513.

    Article  PubMed  Google Scholar 

  38. Giard,’ DJ, Thilly WG, Wang DIC, Levine2 DW. Virus Production with a Newly Developed Microcarrier System. Applied and environmental microbiology. 1977. http://aem.asm.org/. Accessed 16 Dec 2019.

  39. Sinskey AJ, Fleischaker RJ, Tyo MA, Giard DJ, Wang DIC. Production of cell-derived products: virus and interferon. Ann N Y Acad Sci. 1981;369(1):47–59. https://doi.org/10.1111/j.1749-6632.1981.tb14176.x.

    Article  CAS  PubMed  Google Scholar 

  40. YekrangSafakar A, Acun A, Choi J-W, Song E, Zorlutuna P, Park K. Hollow microcarriers for large-scale expansion of anchorage-dependent cells in a stirred bioreactor. Biotechnol Bioeng. 2018;115(7):1717–28. https://doi.org/10.1002/bit.26601.

    Article  CAS  PubMed  Google Scholar 

  41. Tavassoli H, Alhosseini SN, Tay A, Chan PPY, Weng Oh SK, Warkiani ME. Large-scale production of stem cells utilizing microcarriers: a biomaterials engineering perspective from academic research to commercialized products. Biomaterials. 2018;181:333–46.

    Article  CAS  PubMed  Google Scholar 

  42. Rafiq QA, Brosnan KM, Coopman K, Nienow AW, Hewitt CJ. Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor. Biotechnol Lett. 2013;35(8):1233–45. https://dspace.lboro.ac.uk/2134/14778. Accessed 2 Jan 2020.

  43. Malda J, Frondoza CG. Microcarriers in the engineering of cartilage and bone. Trends Biotechnol. 2006;24:299–304.

    Article  CAS  PubMed  Google Scholar 

  44. Yourek G, McCormick SM, Mao JJ, Reilly GC. Shear stress induces osteogenic differentiation of human mesenchymal stem cells. Regen Med. 2010;5(5):713–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Li C, Qian Y, Zhao S, Yin Y, Li J. Alginate/PEG based microcarriers with cleavable crosslinkage for expansion and non-invasive harvest of human umbilical cord blood mesenchymal stem cells. Mater Sci Eng C. 2016;64:43–53. https://www.sciencedirect.com/science/article/pii/S092849311630265X?via%3Dihub. Accessed 11 Aug 2019.

  46. Ferrari C, Balandras F, Guedon E, Olmos E, Chevalot I, Marc A. Limiting cell aggregation during mesenchymal stem cell expansion on microcarriers. Biotechnol Prog. 2012;28(3):780–7.

    Article  CAS  PubMed  Google Scholar 

  47. Hewitt CJ, Lee K, Nienow AW, Thomas RJ, Smith M, Thomas CR. Expansion of human mesenchymal stem cells on microcarriers. Biotechnol Lett. 2011;33(11):2325–35.

    Article  CAS  PubMed  Google Scholar 

  48. Eibes G, dos Santos F, Andrade PZ, Boura JS, Abecasis MMA, da Silva CL, et al. Maximizing the ex vivo expansion of human mesenchymal stem cells using a microcarrier-based stirred culture system. J Biotechnol. 2010;146(4):194–7.

    Article  CAS  PubMed  Google Scholar 

  49. Healthcare G. Microcarrier cell culture: Principles and methods. GE Healthcare/Amersham. 2005. http://www.gelifesciences.co.kr/wp-content/uploads/2016/07/023.8_Microcarrier-Cell-Culture.pdf. Accessed 11 Jan 2020.

  50. Nienow AW, Rafiq QA, Coopman K, Hewitt CJ. A potentially scalable method for the harvesting of hMSCs from microcarriers. Biochem Eng J. 2014;15(85):79–88.

    Article  CAS  Google Scholar 

  51. Chui CY, Odeleye A, Nguyen L, Kasoju N, Soliman E, Ye H. Electrosprayed genipin cross-linked alginate–chitosan microcarriers for ex vivo expansion of mesenchymal stem cells. J Biomed Mater Res - Part A. 2019;107(1):122–33.

    Article  CAS  Google Scholar 

  52. Gröhn P, Klöck G, Zimmermann U. Collagen-coated Ba2 + -alginate microcarriers for the culture of anchorage-dependent mammalian cells. Biotechniques. 1997;22(5):970–5.

    Article  PubMed  Google Scholar 

  53. Heathman TRJ, Stolzing A, Fabian C, Rafiq QA, Coopman K, Nienow AW, et al. Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate. Cytotherapy. 2016;18(4):523–35. https://linkinghub.elsevier.com/retrieve/pii/S1465324916000189. Accessed 11 Aug 2019 .

  54. Nienow AW, Hewitt CJ, Heathman TRJ, Glyn VAM, Fonte GN, Hanga MP, et al. Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms. Biochem Eng J. 2016;108:24–9. https://www.sciencedirect.com/science/article/pii/S1369703X15300309. Accessed 11 Aug 2019.

  55. Lakhkar NJ, Day R, Kim H-W, Ludka K, Mordan NJ, Salih V, et al. Titanium phosphate glass microcarriers induce enhanced osteogenic cell proliferation and human mesenchymal stem cell protein expression. J Tissue Eng. 2015;6:204173141561774. https://doi.org/10.1177/2041731415617741.

    Article  CAS  Google Scholar 

  56. Song K, Yang Y, Wu S, Zhang Y, Feng S, Wang H, et al. In vitro culture and harvest of BMMSCs on the surface of a novel thermosensitive glass microcarrier. Mater Sci Eng C. 2016;58:324–30. https://www.sciencedirect.com/science/article/pii/S0928493115302988?via%3Dihub. Accessed 12 Aug 2019.

  57. Tanimowo Aiyelabegan H, Ebadi M, AliKardar G, Lotfibakhshaiesh N, Abedin Dorkoosh F, EbrahimiBarough S, et al. k-Casein upregulates osteogenic differentiation on bone marrow mesenchymal stem cells cultured on agarose microcarriers. Int J Polym Mater Polym Biomater. 2019;69(6):373–80.

    Article  CAS  Google Scholar 

  58. Dias AD, Elicson JM, Murphy WL. Microcarriers with Synthetic Hydrogel Surfaces for Stem Cell Expansion. Adv Healthc Mater. 2017;6(16):1700072. https://doi.org/10.1002/adhm.201700072.

    Article  CAS  Google Scholar 

  59. Krutty JD, Dias AD, Yun J, Murphy WL, Gopalan P. Synthetic, chemically defined polymer-coated microcarriers for the expansion of human mesenchymal stem cells. Macromol Biosci. 2019;19(2):1800299.

    Article  CAS  Google Scholar 

  60. Gupta P, Hall GN, Geris L, Luyten FP, Papantoniou I. Human platelet lysate improves bone forming potential of human progenitor cells expanded in microcarrier-based dynamic culture. Stem Cells Transl Med. 2019;8(8):810–21.

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Rafiq QA, Hanga MP, Heathman TRJ, Coopman K, Nienow AW, Williams DJ, et al. Process development of human multipotent stromal cell microcarrier culture using an automated high-throughput microbioreactor. Biotechnol Bioeng. 2017;114(10):2253–66. https://doi.org/10.1002/bit.26359.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Yuan X, Tsai A-C, Farrance I, Rowley J, Ma T. Aggregation of culture expanded human mesenchymal stem cells in microcarrier-based bioreactor. Biochem Eng J. 2018;131:39.

    Article  CAS  PubMed  Google Scholar 

  63. Heathman TRJ, Nienow AW, Rafiq QA, Coopman K, Kara B, Hewitt CJ. Agitation and aeration of stirred-bioreactors for the microcarrier culture of human mesenchymal stem cells and potential implications for large-scale bioprocess development. Biochem Eng J. 2018;136:9–17. https://doi.org/10.1016/j.bej.2018.04.011.

    Article  CAS  Google Scholar 

  64. Lin YM, Lim JFY, Lee J, Choolani M, Chan JKY, Reuveny S, et al. Expansion in microcarrier-spinner cultures improves the chondrogenic potential of human early mesenchymal stromal cells. Cytotherapy. 2016;18(6):740–53.

    Article  CAS  PubMed  Google Scholar 

  65. Zhang S, Zhou M, Ye Z, Zhou Y, Tan W-S. Fabrication of viable and functional pre-vascularized modular bone tissues by coculturing MSCs and HUVECs on microcarriers in spinner flasks. Biotechnol J. 2017;12(8):1700008. https://doi.org/10.1002/biot.201700008.

    Article  CAS  Google Scholar 

  66. De Fusco C, Messina A, Monda V, Viggiano E, Moscatelli F, Valenzano A, et al. Osteopontin: relation between adipose tissue and bone homeostasis. Stem Cells Int. 2017;2017:4045238.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. Poon B, Kha T, Tran S, Dass CR. Bone morphogenetic protein-2 and bone therapy: successes and pitfalls. J Pharm Pharmacol. 2016;68(2):139–47. https://doi.org/10.1111/jphp.12506.

    Article  CAS  PubMed  Google Scholar 

  68. Ota M, Takagaki K, Takaoka S, Tanemura H, Urushihata N. A new method to confirm the absence of human and animal serum in mesenchymal stem cell culture media. Int J Med Sci. 2019;16(8):1102–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Lin YM, Lee J, Lim JFY, Choolani M, Chan JKY, Reuveny S, et al. Critical attributes of human early mesenchymal stromal cell-laden microcarrier constructs for improved chondrogenic differentiation. Stem Cell Res Ther. 2017;8(1):93. https://doi.org/10.1186/s13287-017-0538-x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Takahashi I, Sato K, Mera H, Wakitani S, Takagi M. Effects of agitation rate on aggregation during beads-to-beads subcultivation of microcarrier culture of human mesenchymal stem cells. Cytotechnology. 2017;69(3):503–9.

    Article  CAS  PubMed  Google Scholar 

  71. Zhang S, Zhou M, Ye Z, Zhou Y, Tan WS. Fabrication of viable and functional pre-vascularized modular bone tissues by coculturing MSCs and HUVECs on microcarriers in spinner flasks. Biotechnol J. 2017;12(8):1–28.

    Article  CAS  Google Scholar 

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Funding

The research was performed with the financial support of Universiti Kebangsaan Malaysia (FF-2019-448; FF-2019-448/1).

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  1. Tissue Engineering Centre, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, Cheras, 56000, Kuala Lumpur, Malaysia

    Benson Koh, Nadiah Sulaiman, Mh Busra Fauzi, Jia Xian Law, Min Hwei Ng, Ruszymah Bt Hj Idrus & Muhammad Dain Yazid

  2. Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, Cheras, 56000, Kuala Lumpur, Malaysia

    Ruszymah Bt Hj Idrus

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  1. Benson Koh
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Contributions

MDY, RBHI, MHN, JXL and MBF conceived of the presented idea. MDY and BK developed the theory followed by performing the data extractions. MDY and NS verified the data extraction methods and data extracted. MDY supervised the findings of this work. All authors discussed the results and contributed to the final manuscript. All authors read and approved the final manuscript.

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Correspondence to Muhammad Dain Yazid.

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Koh, B., Sulaiman, N., Fauzi, M.B. et al. Three dimensional microcarrier system in mesenchymal stem cell culture: a systematic review. Cell Biosci 10, 75 (2020). https://doi.org/10.1186/s13578-020-00438-8

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