||百姓网公众号微信扫码关注百姓网小程序微信扫扫立即体验扫码下载手机客户端免费抢油卡、红包、电影票您正在浏览信息，点击查看更多服务苏州沧浪回收二手苹果X三星回收魅族华为vivo手机&服务内容：服务范围：所在地：本地上门服务联系人：杜经理联系：(上海)联系时，请一定说明在百姓网看到的，谢谢！见面最安全，发现问题请举报其他联系：x微信号: 1.手机系列：华为、iphone、三星、索尼、HT??C、小米、 oppo、步步高、联想、魅族、黑莓、LG等魅族等手机；2.电脑系列：ipad平板电脑：ipad1ipad2ipad3ipad4ipad5；苹果macbookair系列超薄笔记本电脑,Macbookpro系列笔记本电脑,苹果Mac一体大屏机,苹果高端液晶显示器；,touchipod等一系列苹果产品专业高价回收苹果笔记本苹果一体机各种品牌台式机,SONY（索尼）、HP（惠普）、LG、Thinkpad、戴尔、东芝、方正、富士通、华硕、联想、明基、三星、松下、ACER（宏基）等1.手机系列：华为、iphone、三星、索尼、HT&&&&C、小米、 oppo、步步高、联想、魅族、黑莓、LG等魅族等手机；2.电脑系列：ipad平板电脑：ipad1ipad2ipad3ipad4ipad5；苹果macbookair系列超薄笔记本电脑,Macbookpro系列笔记本电脑,苹果Mac一体大屏机,苹果高端液晶显示器；,touchipod等一系列苹果产品专业高价回收苹果笔记本苹果一体机各种品牌台式机,SONY（索尼）、HP（惠普）、LG、Thinkpad、戴尔、东芝、方正、富士通、华硕、联想、明基、三星、松下、ACER（宏基）等百姓网提醒您：1）接受服务前请仔细核验对方经营资质，勿信夸张宣传和承诺&
2）任何要求预付定金或付款至个人账号的行为，均可能存在诈骗风险，请提高警惕。小贴士：本页信息由用户及第三方发布，真实性、合法性由发布人负责。详情请阅读
(window.slotbydup = window.slotbydup || []).push({
container: s,
id: cpro_id,
scale: '20.3',
display: 'inlay-fix'
1/6显示照片列表信息设置为“搞定了！”状态后，其他用户将无法查看您的联系方式。您确认搞定了这条信息吗？重新发布后可使用“刷新”将发布时间更新为最新时间，并将信息排到第一页。商户推广合作加盟服务支持合作伙伴|&| 沪公网安备16号4&G:86&GM:145
描述：请填写描述手机号：请填写手机号请填写手机号上传图片：打开微信，扫一扫右侧二维码，即可完成绑定 -->绑定后，您可以：1. 立即在手机上收到用户给您的留言2. 使用手机快速完成付费推广的续费动作3. 第一时间了解到百姓网付费推广最新的促销活动，以及享受微信端独特的促销活动4. 更快速地将信息通过微信分享给好友、同事、朋友圈5. 如果您是招聘类目用户，还能够第一时间接收到新简历通知下载APP无需登录实时接受私信提醒，联系更便捷！或点击下方先登录再进入私信联系安卓版下载二维码下载
类型：版本：v1.0.3大小：10M更新：语言：简体等级：平台：安卓
16M / v3.1.0
58M / v10.1.20.568
15M / v1.0.6
4.6M / v9.27
3.2M / v3.7.0.0.0.9
7.0M / v5.9.6
应用说明相关下载相关阅读网友评论下载地址
vivo挖矿安卓版是一款由vivo推出的区块链平台应用，用户可以在该平台上使用数据，浏览等等获得相应的虚拟货币，从而兑换现金收益，操作简单，功能强大，快来下载体验下吧!软件介绍vivo区块链挖矿是一款由vivo推出的区块链平台应用，使用数据，浏览新闻等可获得相应的虚拟货币，从而兑换现金收益软件功能1、行业快讯：及时推送区块链行业要闻及市场行情报价;2、行业快讯：及时推送区块链行业要闻及市场行情报价;3、新闻：独家报道、新闻资讯、深度解读、行业、金色讲堂、金色学院多栏目为区块链行业提供媒体服务和具参考价值的行业信息，让用户快速了解行业变化，为决策提供参考;4、用户至上：限价、市价、三种交易模式，提现快速到账，实时到账，7x24 小时中英双语客服服务;5、专业：创下日交易量纪录501万枚，比特币人民币、美元市场多币种交易，业内专业级操盘工具“闪电手”;6、：超过10年风控经验团队，全方位可定制的安全体系，98%比特币资产存储多重签名冷;相关新闻糖果是从去年逐渐展露头角的一家智能手机品牌，率先推出“创世版区块链手机”，这应该也是全球首款与区块链概念融合的智能手机产品。手机正面采用5.99英寸的全面屏比例设计，背面是一块海军蓝颜色的背板，前后均采用广角双摄方案，同时高达6400万。
vivo区块链挖矿安卓版应用截图
vivo区块链挖矿安卓版相关版本
vivo区块链挖矿安卓版相关文章
vivo区块链挖矿安卓版多平台下载
vivo区块链挖矿安卓版 v1.0.3
58M / v10.1.20.568
9.9M / v4.1.0
11M / v3.5.8
27.5M / v9.40.03
25.6M / v3.0
31.1M / v4.12.0
vivo区块链挖矿安卓版 v1.0.3来自 vivo X7Plus
到底什么怎么样的？
来自 社区电脑版
好久都没你消息了
来自 vivo X6S A
【X7评测】1600万柔光自拍，为自拍而生
【X7美图】有了“你”就有了光，自拍神器vivo X7｜图赏
【X7美图】静待邂逅，vivo X7图赏
【X7Plus美图】更美的大屏手机 vivo X7Plus「图赏」
【X7评测】夏日·乐悠悠｜X7摄影美图系列
24小时全国服务热线
400-678-9688
公众号：vivo智能手机
生活号：vivo智能手机
公众号：vivo智能手机
生活号：vivo智能手机
保存二维码安卓版下载二维码下载
类型：版本：v1.5.9大小：264M更新：语言：简体等级：平台：安卓
216.7M / v3.3.35
274.1M / v1.6.6
651M / v1.0.2
580.4M / v1.1.124
250.3M / v4.9
233M / v1.0.25
应用说明相关下载相关阅读网友评论下载地址
剧情以诙谐风趣为主。一开始主角仿佛梦中，被一声声温柔急促的女声唤醒。熟悉而又幽默的剧情脚本让大家似乎重回《放开那》时的场景游戏介绍《》与放三最大的改变就是操作模式从竖版变成了。为此，玩家们也分成两派，一方觉得人物加入了效果和3D建模，竖版无法展示出来；一方觉得横版不符合操作习惯，竖版更加轻松。游戏攻略2四大阵营主公实力对比分析：吴国孙权孙权半输出型的辅助武将，有点偏向于肉盾。技能是随机对敌方3人造成155%攻击的伤害，并给已方全体附加20%免伤2回合，虽然在输出上表现平凡，但能为队友提供免伤效果，如果把他当作为一个辅助型的肉盾还是非常不错的。蜀国刘备：刘备可以说是一位半输出型的辅助奶妈，有着一定的输出能力，也有着一定的治疗能力。技能是对敌方随机3人造成155%攻击的伤害，并给全体附加持续治疗能力。不管是在输出上还是治疗效果上都表现一般，但在持久战中却能为队友提供很强的续航能力。魏国曹操：曹操又是一个半输出型辅助武将，说白了就是给队友增加百分比的攻击力。技能是对敌方全体造成95%的攻击伤害，并给已方全体附加20%伤害2回合。在几个主公之中算是唯一一个全体攻击的武将，虽然伤害输出过低，但附加伤害尚可，如果用在输出阵容中还是不错的。辅助型主公：董卓董卓光看那如火似焰的眼睛就知道属于暴力份子了，能给敌方造成一定的伤害同时还能增加全体队友的暴击。技能是对敌方横排造成155%攻击的伤害，并给已方全体附加20%暴击2回合。如果用在输出阵容中真的是非常。
放开那三国2vivo版游戏截图
放开那三国2vivo版相关版本
放开那三国2vivo版相关文章
放开那三国2vivo版多平台下载
354M / v1.0
188M / v1.200.4036
651M / v1.0.2
623.6M / v1.0.34
152M / v3.28.2
864.6M / v1.0.31
放开那三国2vivo版 v1.5.9Stem Cells International
Indexed in Science Citation Index Expanded
Journal MenuSpecial Issues Menu
Subscribe toTable of Contents Alerts
Table of Contents Alerts
To receive news and publication updates for Stem Cells International, enter your email address in the box below.
Stem Cells InternationalVolume ), Article ID
pagesReview ArticleEx Vivo Expansion of Human Mesenchymal Stem Cells in Defined Serum-Free Media,1 ,1 ,2 and 11Pharmaceutical Production Research Facility (PPRF), Schulich School of Engineering, University of Calgary, Calgary, AB, Canada T2N 1N42Department of Surgery, McGill University, Montreal, QC, Canada H3G 1A4Received 23 January 2012; Accepted 31 January 2012Academic Editor: Selim Kuçi Copyright (C) 2012 Sunghoon Jung et al. This is an open access article distributed under the , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Human mesenchymal stem cells (hMSCs) are presently being evaluated for their therapeutic potential in clinical studies to treat various diseases, disorders, and injuries. To date, early-phase studies have indicated that the use of both autologous and allogeneic hMSC however, efficacy has not been demonstrated in recent late-stage clinical trials. Optimized cell bioprocessing protocols may enhance the efficacy as well as safety of hMSC therapeutics. Classical media used for generating hMSCs are typically supplemented with ill-defined supplements such as fetal bovine serum (FBS) or human-sourced alternatives. Ideally, culture media are desired to have well-defined serum-free formulations that support the efficient production of hMSCs while maintaining their therapeutic and differentiation capacity. Towards this objective, we review here current cell culture media for hMSCs and discuss medium development strategies.1. IntroductionHuman mesenchymal stem cells (hMSCs), also referred to as mesenchymal stromal cells [], demonstrate regenerative properties and multipotentiality, and thus have been proposed as a potential candidate for cell therapies and tissue engineering. Clinical studies employing hMSCs derived from different sources have been initiated for the treatment of several diseases and injuries such as myocardial infarction, osteogenesis imperfecta, graft-versus-host disease (GVHD), and Crohn’s disease, spinal cord injury, multiple sclerosis, and diabetes (). Early-phase studies with thousands of patients have indicated that the use of both autologous and allogeneic hMSC however, efficacy has not been demonstrated in recent late-stage clinical trials []. In general, clinical protocols employ cell culture technologies by which a small fraction of primary hMSCs are isolated from a selected tissue source and expanded for multiple passages in order to generate a clinically relevant number of cells. Consequently, once the tissue source of hMSCs is determined for an intended clinical application, the safety and efficacy of cell therapeutics produced may be significantly influenced by cell bioprocessing protocols []. As a consequence, developing robust production processes by optimizing culture variables is critical to efficiently and consistently generate hMSCs that retain desired regenerative and differentiation properties while minimizing any potential risks.Cell culture variables include medium formulation (basal media and supplements), culture surface substrate, cell seeding density, physiochemical environment (dissolved oxygen and carbon dioxide concentrations, temperature, pH, osmolality, and buffer system), along with subculture protocols. In particular, the development of well-formulated culture media for both the isolation and expansion of hMSCs is imperative, but has been recognized as an extremely difficult process due to the high complexity of media formulations. Herein, we review various types of media that are currently used for clinical studies or under evaluation, along with the biological characteristics and ex vivo expansion procedures for hMSCs. It is clear that defined media optimized for hMSC isolation and expansion would greatly facilitate the development of robust, clinically acceptable bioprocesses for reproducibly generating quality-assured cells. Although several serum-free formulations have recently been developed, the performance of most media seems to be suboptimal. Identifying critical factors and their concentrations towards designing an ideally formulated, chemically defined serum-free medium should be carried out using rational and systematic approaches. Hence, in the second part of this paper, we discuss crucial strategies and important decisions needed for serum-free medium development.2. Mesenchymal Stem Cells2.1. What Is an MSC?Friedenstein and colleagues first reported a small fraction of cells in bone marrow (BM) attached to and proliferated on tissue culture substrates and that these cells were able to differentiate into multiple cell types such as adipocytes, osteoblasts, and chondrocytes both in vitro and in vivo [reviewed in []]. These cells were fibroblastic spindle-shaped and readily generated single-cell-derived colonies and were originally referred to as colony-forming unit-fibroblasts (CFU-F). Later, these cells were demonstrated by many investigators to be heterogeneous populations of nonhematopoietic adult stem/progenitor cell-like cells residing in marrow stroma and, thus, were called marrow stromal cells, mesenchymal stem cells, or multipotent mesenchymal stromal cells [, ]. In addition to BM, similar MSC-like cells have also been shown to be present in most tissues, including adipose tissue (AT), synovial membranes, bone, skin, pancreas, blood, fetal liver, lung, and umbilical cord blood (UCB) [, ].2.2. Characteristics of hMSCsBM has been the traditional source of hMSCs for basic research and therapeutic use because BM harvest is a routine and safe procedure. Therefore, the characteristics of ex vivo expanded hMSCs described below mostly represent BM-derived hMSCs unless otherwise stated.2.2.1. MorphologyTypically, hMSCs isolated and expanded in classical FBS-containing media are mostly spindle-shaped (or fusiform) and cuboidal fibroblast-like cells. More specifically, Prockop and colleagues demonstrated that hMSCs undergo a time-dependent morphological transition from thin (small), spindle-shaped cells (considered stem cells or early progenitors) to wider (larger), spindle-shaped cells (looked like more mature cells) when cells are plated at 1 to 1,000 cells/cm2 []. They further showed that the small, spindle-shaped cells proliferate more rapidly and have a higher level of multipotentiality, compared to the slowly replicating large cells, which have lost most of their multipotentiality but can still differentiate into a lineage (e.g., osteogenic) as a default pathway. The morphology (and size) of hMSCs may also be dependent upon culture conditions (e.g., growth media, culture surface). For example, hMSCs expanded in bFGF-supplemented media were smaller and proliferated more rapidly, compared to those in bFGF-lacking control conditions []. Culture surfaces (e.g., treated with Matrigel) might also affect the morphology [].2.2.2. Growth and Adherence CharacteristicshMSCs are anchorage-dependent cells, which attach to a plastic surface, spread out, and grow, when maintained in standard culture conditions (e.g., DMEM supplemented with 10% FBS). The initial growth of hMSCs in primary BM cell culture on a plastic surface is characterized by the formation of single-cell-derived colonies, when the cells are plated at appropriate numbers. The cells in colonies generated in the primary culture can typically be subcultured through multiple passages at various plating densities. In general, hMSCs have great propensity for expansion in culture, although their proliferation potential is highly variable, depending on many aspects such as donor age, tissue source, and culture conditions. For example, Sekiya et al. demonstrated that hMSCs proliferate more rapidly when passaged by plating the cells at low densities (e.g., 10&#x cells/cm2, compared to 1,000&#x cells/cm2) []. hMSC proliferation is also highly variable depending on growth media [].2.2.3. ImmunophenotypeCurrently, no prospective markers exclusively defining hMSCs are available. In general, hMSCs are negative for hematopoietic surface markers including Cluster of Differentiation (CD) 34, CD45, CD14, CD11b, CD19, CD79α, CD31, CD133 and positive for CD63, CD105, CD166, CD54, CD55, CD13, CD44, CD73, and CD90 [, ]. However, differences exist among the studies reporting the surface marker characteristics, which may be explained by variations in culture methods and/or differentiation stage of the cells [].2.2.4. Multilineage Differentiation PotentialhMSCs have at least trilineage differentiation potential in vitro (i.e., the ability to differentiate into bone, cartilage and fat upon proper induction conditions). This is the most well-established characteristic of hMSCs and thus is considered the hallmark of these cells []. It has also been observed that hMSCs could give rise to other mesodermal cells and nonmesodermal cell types, such as neuron-like and endoderm-like cells [].2.2.5. Minimal Criteria for Defining hMSCsStandard culture protocols for the isolation and expansion of hMSCs have not been well established, in particular for growth medium. Therefore, different laboratories often use various methods of isolation/expansion and also different approaches to characterize the cells. This makes it difficult to compare and contrast the outcomes from various investigators. To minimize the variations, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy has proposed minimal criteria to define hMSC populations. These include the following: (i) hMSCs must be plastic-adherent when maintained in classica (ii) hMSCs must express high levels (≥95% positive) of CD105, CD73, and CD90 and lack expression (≤2% positive) of CD45, CD34, CD14, or CD11b, CD79α or CD19, and HLA-DR (unless stimulated by interferon-γ) (iii) hMSCs must differentiate into osteoblasts, adipocytes, and chondroblasts under specific in vitro differentiation conditions [].2.3. Potential Therapeutic Properties of hMSCshMSCs show various properties that could be important in therapeutic applications. Indeed, some of these functions are currently being exploited in clinical trials with great promise.2.3.1. MultipotentialityAs described earlier, hMSCs can differentiate into distinctive mesenchymal phenotypes, and thus they have been used to reconstruct damaged tissues upon transplantation in association with scaffolds. In addition, due to their differentiation potential into nonmesodermal cell types, hMSCs cells have also been proposed for replacement therapies to treat various diseases and disorders such as neuronal diseases and diabetes [, ].2.3.2. Tropism for Sites of DiseasehMSCs appear to be capable of homing to sites of disease or damage. It has been reported that hMSCs systematically infused into diabetic mice migrated to damaged sites (i.e., pancreatic islets and renal glomeruli) and contributed to the repair of tissue [] by certain mechanism(s) yet unidentified.2.3.3. Secretion of Bioactive FactorshMSCs inherently synthesize and secrete a broad range of bioactive agents such as cytokines and growth factors []. This intrinsic secretory activity of hMSCs may significantly contribute to tissue repair or regeneration, presumably by establishing a regenerative microenvironment at sites of tissue injury or damage []. Originally, therapeutic effects observed with the use of hMSCs were thought to be due to their transdifferentiation (i.e., differentiation into nonmesodermal cell types) potential. However, these beneficial effects were often demonstrated without evidence for the engraftment and transdifferentiation of transplanted hMSCs in animal model studies. Therefore, these indirect, secretory functions of hMSCs have been proposed as an alternative mechanism explaining the therapeutic effects, and importantly, these characteristics have generated clinical interest to use undifferentiated hMSCs for various applications such as repair or regeneration of damaged tissues [].2.3.4. ImmunomodulationhMSCs also display immune regulatory properties that might represent a critical role in the therapeutic application of these cells []. In vitro studies using hMSCs demonstrated that these cells suppress the proliferation of T cells. Further, it was also revealed that hMSCs inhibit the differentiation and maturation of dendritic cells (DCs) and decrease the production of inflammatory cytokines by various immune cell populations []. DCs are the most potent antigen-presenting cells, which specialize in antigen uptake, transport, and presentation and have the unique capacity to stimulate na&#ve and memory T cells []. In addition to the in vitro effects, it has been seen in animal model studies that hMSCs may also display immunosuppressive capacities in vivo. For example, hMSCs facilitated engraftment of hematopoietic stem cells and prolonged skin allograft survival []. Further, it has been demonstrated that the use of hMSCs reversed severe acute GVHD []. However, while in vitro results consistently show the immunosuppressive capacity of hMSCs, studies in animals and humans suggest that hMSCs are less effective in producing systemic immunosuppression in vivo []. Therefore, further studies using standardized hMSC populations are urgently needed to verify their in vivo immunosuppressive potential and to define the optimal conditions for the use of hMSCs as immunotherapy.2.3.5. Immune Privileged or Hypoimmunogenic PropertyWhen undifferentiated hMSCs were transplanted into recipients in preclinical and clinical trials, these cells produced various cytokines and growth factors and had an ability to modify the response of immune cells. Another important observation is that hMSCs escape immune recognition or at least possess a hypoimmunogenic character upon allogeneic transplantation []. Indeed, clinical studies showed that hMSCs evoke little or no immune reactivity in allogeneic recipients []. This indicates that, in addition to the transplantation of autologous cells to patients to minimize the risk of immune response, allogeneic hMSCs could also be safely used. If this is the case, the use of allogeneic hMSCs has an important advantage in that the culture-expanded cells could be considered as an “off the shelf” therapeutic product. Indeed, clinical studies using allogeneic, as well as autologous, hMSCs from BM have been initiated for the treatment of several diseases and injuries such as osteogenesis imperfecta, GVHD, leukemia, myocardial infarcts, Crohn’s Disease, cartilage and meniscus injury, stroke, and spinal cord injury [, ].2.4. Safety of Using hMSCsA large body of safety data has been gained from the use of hMSCs in various clinical applications including the treatment of GVHD and the facilitation of BM engraftment []. That is, until present, only few adverse effects attributed to hMSC transplantation have been reported []. This has facilitated the rapid translation of basic research into clinical trials. However, there are some obvious issues that need to be addressed before the wide implementation of clinical trials using hMSCs.2.4.1. Tumor FormationIn general, it is considered that hMSCs can be safely cultured in vitro with no risk of malignant spontaneous transformation []. Stenderup and colleagues cultured several strains of hMSCs from BM at various ages (i.e., aged 18–81 years) until the cells reached their maximal life span without any evidence of transformation []. Further, there have been no reports with human trials demonstrating the formation of tumors by culture-expanded hMSCs []. Nonetheless, a potential risk for spontaneous transformation associated with hMSC proliferation in vitro, in particular after long-term culture, cannot be ruled out. The transplantation of primitive stem/progenitor cells with considerable proliferative potential can raise the possibility of tumor formation, and any ex vivo manipulation will increase the chances of transformation []. It has been recommended by the Food and Drug Administration (FDA) that “minimally manipulated” cells be used for human clinical trials. In this regard, attempts are being made to develop an efficient production system to produce clinically relevant numbers of hMSCs at relative shorter periods of time with lower passage numbers [].2.4.2. Promotion of Tumor Growth and DevelopmentA potential risk of treatment with hMSCs can paradoxically arise from the fact that these cells are capable of suppressing various immune cells, which may promote tumor growth and metastasis. The role of hMSCs in tumors is controversial: some studies have demonstrated enhancement of tumor growth and metastasis, while others have shown no apparent effect or inhibition of tumor growth with the use of hMSCs [].2.4.3. Immune ResponseAlthough hMSCs themselves appear to escape immune recognition, those “cultured in medium containing FBS” can produce immune reactions in patients receiving repeated administrations of these cells []. Conventionally, the optimal conditions for hMSC expansion require media supplemented with FBS at a concentration of 10–20%, which corresponds to approximately 5–10 mg FBS proteins/mL of medium. Spees et al. demonstrated that, when hMSCs were cultured in medium containing 20% FBS and harvested, 7–30 mg of FBS proteins were still associated with a standard preparation of 100 million hMSCs, a dosage that probably will be needed for clinical therapies []. Thus, immunological reactions caused by medium-derived FBS proteins will be a concern, in particular for certain types of cell therapy involving multiple administrations of hMSCs. The safety issue associated with the use of FBS in culture media will be avoided by developing an alternative culture protocol to produce hMSCs in vitro in the absence of FBS.2.5. Sources of hMSCsAlthough the traditional source of hMSCs is BM, it has been demonstrated that cells displaying similar characteristics with BM-hMSCs can also be derived from other sources including AT, UCB, umbilical cord tissue, placenta, amniotic fluid, liver, lung, pancreas, and muscle [–]. The ideal source of hMSCs for therapeutic use would be one that is readily available and can be expanded in culture rapidly to yield large numbers of cells. In this regard, hMSCs from readily obtainable tissue otherwise discarded, such as AT, may offer a preferable alternative to BM, because the collection of BM is an invasive procedure. Moreover, AT is a source of abundant hMSCs, and the AT-derived hMSCs have shown various potential therapeutic properties both in vitro and in vivo []. UCB, umbilical cord tissue, and placenta also represent attractive sources of hMSCs because these tissues are readily available and the derived cells may contain less genetic abnormalities and greater proliferative capacity than adult tissue-derived hMSCs [–].It is important to note that there is much evidence demonstrating that hMSCs (or similar populations) derived from various sources showed different characteristics in gene expression profile, proliferation and differentiation potential, and functional properties although most of these satisfied the minimal criteria for defining MSCs and, thus, were considered as hMSCs as a whole [–]. For example, studies have shown that AT-hMSCs exhibited in vitro immunomodulatory properties at higher efficiencies, compared to BM-derived counterparts []. Another example can be found from a study comparing the differentiation potential of hMSCs from BM and pancreas into insulin-producing endocrine cells []. This study revealed that hMSCs derived from the pancreas are committed to an endocrine fate and thus have a greater propensity to generate insulin-producing cells compared to BM-hMSCs. Therefore, to select an ideal source of hMSCs for therapeutic use, their functional properties (e.g., differentiation potential, immunomodulation, secretion of bioactive factors) should be critically evaluated in comparison with those from other potential sources, in addition to the availability of tissue and cell proliferation capacity as mentioned earlier.3. Generation of Mesenchymal Stem Cells3.1. hMSC NumberThe frequency of hMSCs in BM is very low. The CFU-F assay is widely used to estimate the number of MSCs in primary BM cells as well as passaged cell populations in culture [, ]. Using this assay, it has been reported that MSCs represent 0.01% to 0.001% of human BM mononuclear cells (MNCs) []. It was also demonstrated that hMSCs comprise approximately 1 in 10,000, 100,000, and 250,000 BM-hMNCs of newborns, teens, and 30 year-olds, respectively, indicating that the hMSC frequency is highly variable with age [].There is a significant difference in the frequency of hMSCs present in other tissues/organs, heavily depending on the source. Kern et al. reported different frequencies of hMSCs in BM, AT, and UCB. In their study, with culture-initiating populations (i.e., MNCs of BM and UCB, and stromal vascular fraction of AT), it was demonstrated that the number of CFU-Fs (i.e., corresponding to hMSCs) calculated at the basis of 1 × 106 initially plated cells was highest for AT (557), followed by BM (83) []. In contrast, the frequency of CFU-Fs in UCB was considerably lower. Similar observations were reported by others [, ].Although the dosage of hMSCs for their optimal use in therapeutic applications is still unclear and should be dependent upon the type of cell therapy, at least 1 to 2 × 106 hMSCs per kg body weight of the adult patient is generally suggested []. Consequently, the number of primary hMSCs, regardless of the sources, is insufficient for research as well as clinical use. Hence, it is necessary to isolate hMSCs and then subsequently expand them for multiple passages on tissue culture substrates in order to generate clinically relevant numbers of cells.3.2. Isolation and Expansion of hMSCs3.2.1. Isolation of hMSCs (Primary Culture)As there are no universal surface markers available for exclusively defining hMSCs, these cells have traditionally been isolated from initial primary cell fractions (e.g., BM MNCs), based on their selective adherence, compared to hematopoietic cells, to plastic surfaces. Therefore, the hMSCs obtained are intrinsically heterogeneous. Importantly, it has been demonstrated that the characteristics of isolated cells are highly dependent upon culture conditions used. For BM cells, either unfractionated whole BM, fractionated MNCs by density gradient, or separated cells by depletion of certain subpopulations are used, with the fractioned MNCs being the most popular. 3.2.2. Expansion of hMSCsAs a large number of hMSCs is needed for their clinical use, cells obtained from the primary culture are further expanded through multiple passages. Typically, hMSCs obtained from young donors can undergo 24–40 population doublings (PD) in culture before they reach senescence, while those from older donors retain reduced proliferative potential []. Similar to other diploid cells, hMSCs grow at a rather constant rate during early passages (typically for the first 2 to 3 weeks) and then with a gradual increase in cell doubling times as the passage number increases until the growth ceases due to senescence []. It is also known that, after the initial culture, hMSCs progressively show loss of multipotentiality [] under classical media and possibly other culture conditions.3.3. Culture Media for hMSCs3.3.1. Classical FBS-Based MediaConventional media used for isolating and expanding hMSCs include supplementation of FBS at 10–20% (v/v). FBS contains a high content of attachment and growth factors as well as nutritional and physiochemical compounds required for cell maintenance and growth. The function and characteristic of serum is further reviewed later in this paper. FBS-based media remain a common standard in generating hMSCs for basic research
however, the use of FBS is not desirable, raising several safety and other concerns. The inherent potential problems associated with the ill-defined FBS and other animal-derived supplements are as follows [–]:(i)risk of contamination associated with harmful pathogens such as viruses, mycoplasma, prions, or unidentified zoonotic agents and transmissions of these contaminants to cells being used for cellular therapy,(ii)high content of xenogeneic proteins that can be associated with cell therapeutics during culture, causing concerns relating to immune reaction in patients, as described earlier,(iii)high degree of batch-to-batch variation causing inconsistency in the generation of quality-assured cells and thus making standardization of the production process difficult,(iv)presence of growth inhibitors, cytotoxic substances, and/or differentiation agents. (Beyond its growth-promoting property, serum may also contain components that are inhibitory for the growth of certain cell types. It has been demonstrated that some cell types cannot be cultured in the presence of serum at its typical concentrations in medium due to its unidentified cytotoxic constituents. It is also well known that serum is toxic at high concentrations for most cell types []),(v)requirement of a set of strict quality controls to minimize the risk of contamination and to select appropriate FBS lots supporting growth of cells while retaining their regenerative and differentiation properties,(vi)interference of unidentified factors on the effect of hormones, growth factors, or other additives under investigation,(vii)limited availability,(viii)ethical issues [].Considered together, despite strict selection and testing for safety and growth-promoting capacity, the use of FBS represents a major obstacle for the wide implementation of hMSC-related therapies.3.3.2. Humanized MediaIn order to alleviate the safety and regulatory concerns raised by the use of animal serum for generating hMSCs, autologous or allogeneic human blood-derived materials, including human serum, plasma, platelet derivatives (e.g., platelet lysate), and cord blood serum, are currently under investigation for their clinical utility as an alternative medium supplement.Human autologous serum has been reported to support hMSC expansion [–]. It would be problematic, however, to acquire amounts sufficient to generate clinically relevant numbers of hMSCs. Moreover, the use of autologous serum may not be applicable for elderly patients as its capacity to support cell growth may decrease with their age. The performance of human allogeneic serum from adult donors is rather controversial because contradictory results have been reported [, –]. Allogeneic human serum from UCB [, ] and placenta [] has also been proposed as potential alternatives to replace FBS because these primitive tissues are a rich source of growth factors [].Attempts have also been made by many investigators to examine the utility of human platelet lysate (hPL), which has been prepared by mechanical disruption or chemical lysis of the platelet membrane [], for the cultivation of hMSCs. Most of the studies reported that the growth factor-enriched allogeneic hPLs have considerable growth-promoting properties for hMSCs while maintaining their differentiation potential and immunomodulatory properties [–]. However, some other studies reported data that showed a reduction of osteogenic or adipogenic differentiation potential when hMSCs were cultured in hPL-based media [, ]. Moreover, a recent report illustrated that, although cell proliferation was greatly enhanced, the use of hPL (supplemented into RMPI 1640 medium) altered the expression of some hMSC surface molecules and led to a decrease in their in vitro immunosuppressive capacity []. This study also showed that the production of prostaglandin E2, which has previously been demonstrated to play a major role in the suppression of immune cells [], was lowered under the use of hPL compared to FBS.Although considered relatively safer than FBS for human therapeutic applications, the use of human-sourced supplements is still a matter of substantial debate, prompting some concerns [, ]. There is a risk that allogeneic human growth supplements may be contaminated with human pathogens that might not be detected by routine screening of blood donors. Moreover, these crude blood derivatives are poorly defined and suffer from batch-to-batch variation, and thus their ability to maintain hMSC growth and therapeutic potentials could be widely variable. In particular, the variability can be a significant hindrance for implementing the clinical-scale production of hMSCs simply because it could make it difficult to obtain cells retaining desired qualities in a consistent and predictable manner, which is crucial for minimizing treatment failures.3.3.3. Defined Serum-Free MediaThe concerns raised from the use of ill-defined serum or human-sourced supplements demonstrate the need for the development of defined serum-free media. While reducing the problems associated with such crude materials, defined serum-free media may further provide additional advantages as follows.(i)Defined formulations designed to support the generation of a population enriched with a desired cell type (i.e., hMSCs) while preventing overgrowth of undesired cells in primary cultures will lead to the production of more homogeneous hMSCs (i.e., more precisely, a population of adherent cells containing a high content of colony-forming multipotent mesenchymal cells). It has been shown that medium formulations greatly affect the frequency and size of colonies in the primary and passaged hMSC culture, and a systematically optimized defined medium for hMSCs led to significant increased colony-formation compared to FBS-based cultures [].(ii)The well-defined nature of the medium would facilitate enhancing cell bioprocessing protocols, which may be crucial for increasing clinical efficacy by producing cells with desired properties. For instance, based on the proposed mechanism that hMSCs exert therapeutic benefits via the secretion of certain soluble molecules, the medium formulations may need to be modified to enhance expression of specific genes to achieve an optimal cytokine profile [].(iii)When ex vivo differentiated cells are desirable for therapeutic use, the transition of an expansion state to a differentiation phase under defined conditions could facilitate the production of such desired cell types in a more favorable and controllable environment. Davani et al. demonstrated that, in an effort to differentiate hMSCs (pancreas-derived) to insulin-expressing cells, the shift of a serum-based expansion condition to a serum-free differentiation condition led to considerable cell death []. In principle, it may be less harsh to cells to switch only key “growth-promoting factor(s)” to “differentiation-inducing factor(s)” while maintaining the base condition.Attempts have been made to develop defined serum-free media for animal or human MSC however, most of them have demonstrated only limited performance [–]. These media formulations were only shown to support cell expansion for single-passage cultures or at slow rates through multiple passages. Moreover, all of these studies used cells which had previously been exposed to serum during the initial isolation/expansion phases. Serum-derived contaminants are probably carried over with the cells when they are placed under serum-free conditions after exposure to serum, and thus, exposure to serum may ultimately limit their therapeutic use.The ideal media should consist of chemically defined constituents that support the attachment and growth of hMSCs primary cultures as well as passaged cultures, while maintaining their therapeutic properties. Towards this objective, our group has recently carried out a study to identify key attachment and growth factors required for both primary and passaged cultures, and this study led to the development of a defined serum-free medium (PPRF-msc6) for hMSC isolation and expansion []. We demonstrated that PPRF-msc6 medium supported the generation of hMSCs from multiple BM samples in a rapid and consistent manner, maintaining their multipotency and hMSC-specific immunophenotype. Furthermore, compared to a classical serum-supplemented media (i.e., DMEM supplemented with prescreened FBS), hMSCs cultured in PPRF-msc6 exhibited numerous advantages from a production standpoint. Specifically, these hMSCs had a greater colony-forming capacity in primary as well as passaged cultures, negligible lag phase and explicit exponential growth, lower population doubling times (21–26 h versus 35–38 h; between passage levels 1 and 10), a greater number of population doublings (62 ± 4 versus 43 ± 2; over a two-month period), and a more homogeneous cell population, which was smaller in size []. Consequently, the sustained production of smaller hMSCs in a rapid manner requires less time and surface area to obtain clinically relevant numbers of hMSCs, while reducing risk of contamination and saving cost and labor. Moreover, from a therapeutic viewpoint, the size of cells to be transplanted into patients could be an important issue because it has been shown in animal-model studies that most of hMSCs grown in FBS-supplemented media were trapped in the lung []. Small hMSCs may offer a significant benefit in transplantation therapies because the small cells may travel through the lung and home to the site of injury or disease at high efficiencies []. Similar to the performance with BM cells, PPRF-msc6 also allowed for the isolation and extended expansion of hMSCs from other sources, including AT and pancreatic tissue samples, more rapidly and efficiently compared to control FBS-supplemented media [our unpublished data]. In view of the “defined” status, PPRF-msc6 contains some serum components, such as insulin, transferrin, serum albumin, and fetuin, which are often associated with traces of other serum constituents, and thus further investigations need to be conducted to refine this medium to a true chemically defined medium by replacing these components with synthetic alternatives. Recently, for instance, we have successfully replaced native insulin with a recombinant insulin without any decrease in the performance of PPRF-msc6 for hMSC culture. The replacement of native transferrin, albumin, and fetuin in this medium with recombinant alternatives or other supplements is currently underway. Nonetheless, PPRF-msc6 represents the most well-defined serum-free formulation to support both the isolation and expansion of hMSCs in the literature to date and a significant step forward for producing hMSC therapeutics. The protocol for PPRF-msc6 preparation has been described in detail [] so that the disclosed formulation or its modifications can be further developed by any interested party.Efforts have also been made to test or modify existing defined medium formulations designed for other stem cell types in order to cultivate hMSCs. Rajala et al. illustrated that a defined, xeno-free medium for human embryonic stem cells (hESCs) allowed, during a single-passage culture, the expansion of hMSCs previously isolated from AT samples in the presence of allogeneic human serum []. This study did not report whether this medium supported the growth of hMSCs in primary culture as well as through multiple passages. Also, Mimura et al. modified a defined hESC medium to promote the expansion of an immortalized genetically modified hMSC line []. The cells grown in their disclosed medium formulation demonstrated differentiation capacity towards osteogenic and adipogenic lineages while displaying a rather different gene expression profile compared to those cultured in FBS-based medium.3.3.4. Commercially Available Media for Expanding hMSCsSeveral commercially available serum-free media have recently been introduced for the expansion of hMSCs [reviewed in []]. StemPro MSC SFM from Invitrogen represents the first commercial serum-free medium that allows the isolation and expansion of hMSCs from BM and has recently been cleared by the FDA as a medical device for clinical trials in the United States (). Agata et al. demonstrated that this medium supported hMSC growth more rapidly at early passages while reaching senescence earlier (at passage 5) with gradually reduced proliferation rate, compared to a control FBS medium []. In addition, although most of the hMSC-specific surface antigens were expressed on both cell populations expanded in the serum-free medium and an FBS-based reference medium, some molecules were expressed in different levels (i.e., CD105 and CD146). Moreover, it appears that both cell populations displayed different levels of stemness as well as different differentiation potential (i.e., cells expanded in serum-free medium exhibited lower ALP activity in noninduced state, but a greater response to osteogenic induction compared to serum-based controls). In summary, this commercial serum-free medium seems to generate hMSCs with different characteristics in comparison with those derived in classical FBS media.Aiming towards the widespread implementation of hMSC-related therapy in later stage of clinical trials, serum-free, xeno-free media for hMSC culture have also been commercialized. However, the formulations of these commercial media are not disclosed, which may restrict their wide utility in hMSC research and clinical studies. The identification of specific medium components allowing for the serum-free isolation and expansion of hMSCs would contribute significantly to the research and therapeutic applications of these cells. Moreover, as medium formulations determine cell characteristics (i.e., growth pattern, gene expression, phenotype, and functional properties), it is important to evaluate carefully each of these commercial media for intended therapeutic applications, preferably in parallel to select the best choice for each specific target.We have recently initiated a study to compare these commercial media along with other existing media. The media under investigation include Mesencult-XF (STEMCELL Technologies), StemPro MSC SFM Xeno-Free (Invitrogen), MSCGM-CD (Lonza), PPRF-msc6, and DMEM +10% FBS (Lonza). Our initial data show that the performance of the commercial hMSC media on the attachment and growth of primary BM-hMSCs is questionable and thus should be open to discussion. Specifically, in a preliminary experiment, the commercial media were evaluated in parallel with PPRF-msc6 and 10% FBS DMEM by plating human BM MNCs into human fibronectin-coated T-25 flasks containing each of the media. Cells were inoculated at 150,000 cells/cm2, and nonadherent cells in each medium were removed after 60 hours with 100% medium change. Thereafter, the adherent cells were allowed to grow with 50% medium replacement every other day, and then stained on day 12. In this experiment, none of the commercial media demonstrated cell growth (Figures (a)–(c)). In contrast, a significant number of well-developed colonies were found in the culture with PPRF-msc6 (Figure (d)). The serum-based control culture with 10% FBS DMEM also resulted in the formation of colonies although most of them were still premature. Lindroos et al. reported that StemPro MSC SFM Xeno-Free medium provided significantly higher proliferation rates of AT MSCs when compared with serum-containing media []. In their study, however, the authors tested the commercial serum-free medium using cells that were previously isolated and expanded in a serum-containing medium (10% human serum), and the ability of StemPro MSC SFM Xeno-Free medium to allow the growth of primary hMSCs was not addressed. Moreover, Hartmann et al. stated that they were not able to culture hMSCs derived from the UC tissue using StemPro MSC SFM Xeno-Free medium without serum []. In contrast, the authors demonstrated that Mesencult-XF medium supported the isolation and expansion of UC-hMSCs without the use of serum. In an attempt to culture hMSCs from BM using a xeno-free protocol (i.e., proprietary Mesencult-XF Attachment Substrate as well as medium), Miwa et al. also showed that the use of Mesencult-XF medium resulted in the growth of primary BM-hMSCs more rapidly than a serum-containing medium [].Figure 1: Cultivation of primary human BM MNCs using different media including three commercial media. Cells were inoculated at 150,000 BM MNCs/cm2 into fibronectin-coated T-25 flasks, each containing 8 mL of Mesencult-XF (a), StemPro MSC SFM Xeno-Free (b), MSCGM-CD (c), and PPRF-msc6 (d), and a classical FBS medium (10% FBS DMEM) (e). After 60 h, nonadherent cells in each medium were removed, and fresh medium was added to the adherent cells (100% medium change). The adherent cells were allowed to grow for additional 10 days with 50% medium change every other day, and then stained with crystal violet to visualize colonies generated.The contradictory data between our work and the literature [, ] regarding Mesencult-XF medium may be due, at least in part, to the use of different substrate materials. Specifically, we used human fibronectin-coated flasks, while Miwa et al. used a proprietary substrate-coating product. In this regard, we plated the nonadherent cells, which were removed from the first medium change during the primary culture with each medium described earlier, into new flasks containing the same medium to allow them to attach to the new substrate and grow. For convenience, here we call these as “secondary” cultures as opposed to their original cultures described in the previous paragraph and Figure . The “secondary” flasks were coated with gelatin (bovine), which is widely used to facilitate the attachment of many types of anchorage-dependent cells to the substrate. In these secondary cultures, we observed that a number of colonies were formed in the culture with Mesencult-XF medium (Figure (a)). Together with the data obtained from the original culture (Figure (a)), this demonstrates the ability of Mesencult-XF medium to allow the growth of primary hMSCs, but implying that the performance of the medium could be improved by manipulating the substrate-coating materials. In contrast, StemPro MSC SFM Xeno-Free medium and MSCGM-CD medium did not allow for colony formation (Figures (b) and (c), resp.). These data suggest that factors required for the isolation of hMSCs from primary cultures seem to be missed in both media. Similar with the study by Hartmann et al. [], we also observed that the addition of low serum (i.e., 2% prescreened FBS) into StemPro MSC SFM Xeno-Free medium (and MSCGM-CD medium) supported the growth of primary BM-hMSCs (data not shown). Based on these observations, therefore, we would argue that it should be desirable to further optimize all the commercial media described here for the serum-free, xeno-free isolation of hMSCs. Regarding PPRF-msc6, beyond the high number of large colonies generated in the original culture (Figure (d)), a number of colonies also appeared in the secondary cultures (Figure (d)), which were initiated with the nonadherent cells that had been normally discarded in our previous work []. In contrast, the FBS-based culture led to the appearance of only a few colonies in its secondary culture (Figure (e)), indicating that the majority of hMSCs obtainable from the use of 10% FBS DMEM attached to the surface of original culture in the presence of known and unknown attachment factors included in serum. Considered together, these data imply that the yield of hMSCs obtained from the primary culture of BM cells with PPRF-msc6 could further be increased by modifying the culture protocols, particularly by identifying optimal attachment factors and/or substrate-coating materials to enhance initial cell attachment efficiencies.Figure 2: Cultivation of nonadherent cell fractions removed from the culture of primary BM MNCs in different media. Nonadherent cells and spent medium removed from each of the flasks, demonstrated in Figure , were replated into a new T-25 flask coated with gelatin containing 4 mL of the fresh medium—that is, Mesencult-XF (a), StemPro MSC SFM Xeno-Free (b), MSCGM-CD (c), PPRF-msc6 (d), and 10% FBS DMEM (e). After 60 h, nonadherent cells and medium in each flask were discarded, and fresh medium was added to the adherent cells (8 mL per flask). The adherent cells were allowed to grow for additional 8 days with 50% medium change every other day and then stained with crystal violet to visualize colonies generated.A commercial medium (mTeSR) with disclosed composition, which was originally developed for the expansion of hESCs, has also been tested for hMSC culture []. Although this defined medium together with human fibronectin-treated substrate allowed the expansion of BM-hMSCs previously isolated using FBS-based medium, it did not support cell growth in primary BM cultures. In addition, when hMSCs were plated into the mTeSR at a very low density for a CFU-F assay, the colonies derived were significantly smaller at lower frequency, compared to a control case with FBS-based medium. Moreover, mTeSR-derived cells demonstrated significantly decreased adipogenic potential. Therefore, further studies should be carried out with this medium to identify factors affecting multipotency as well as growth of hMSCs to make the disclosed formulation viable for research and clinical applications.In summary, although numerous defined hMSC media are commercially available or have been introduced in the literature to support the growth of hMSCs, one must realize that the therapeutically relevant properties of culture-expanded hMSCs could be significantly affected by medium components. Considering the safety and efficacy required to produce hMSC therapeutics for intended clinical applications, it is crucial to compare different media (and their formulations if the recipe is disclosed) and probably further optimize the formulations in a systematic manner. In this regard, the disclosed medium formulations for hMSCs (e.g., those reported in [, , , ]) are best positioned to be further developed by the many investigators interested in therapeutic applications of hMSCs.4. Development of Defined Serum-Free MediaDefined serum-free medium development or optimization for a specific cell type is a very complicated process because multiple variables that affect the maintenance, growth, and characteristics of cells are interrelated. Moreover, designing a new serum-free formulation for anchorage-dependent cells such as hMSCs tends to be more fastidious compared to those grown in suspension culture, as the interaction of cells with the substrate on which they attach and spread prior to growth needs to be understood. Medium development studies should involve rational approaches: (i) to select appropriate factors (e.g., basal medium formulations and growth/attachment proteins) and (ii) to screen them in a stepwise, systematic manner for their effect on cell properties and growth.4.1. Cell Culture RequirementsAn understanding of the requirements for successful cell culture is a prerequisite for designing a rational strategy towards the efficient development/optimization of a new serum-free medium. In addition, since anchorage-dependent hMSCs normally require serum, namely, some serum components yet unidentified but responsible for their attachment, spread, and growth, it is particularly crucial to understand the functions that serum serve in cell culture in order to identify such components. Hence, specific requirements of nutrients and nonnutrient elements for cell culture are briefly reviewed below with a special emphasis on the constituents and functions of serum.4.1.1. NutrientsNutrients refer to chemical substances that are taken into cells and utilized as substrates in energy metabolism or biosynthesis, as catalysts in those processes, or as structural components of cellular organelles. The nutrients are divided into organic nutrients, inorganic salts, and trace elements, and the organic nutrients are further subdivided into amino acids, carbohydrates, lipids, vitamins, and others []. Generally, the nutrients are considered as the “defined” portion of culture media. In addition to the “nutrient” roles, the nutrients also have regulatory functions. It has been demonstrated that these nutrients could represent the only requirements for in vitro growth of certain transformed cell lines []; however, more fastidious nontransformed normal cells typically require additional growth-promoting supplements for their growth in culture.Amino acids represent essential elements of media as building blocks for protein synthesis. In addition, certain amino acids have other key roles in multiplication of cells in culture, especially under serum-free conditions. In particular, glutamine appears to play major roles in many metabolic pathways, and thus an adequate extracellular concentration of glutamine is typically needed in cell culture media. It is common to add 2–4 mM of glutamine to hMSC media. It is important to note that glutamine is labile under cell culture conditions, and thus the amount of glutamine for hMSC culture should be determined considering both the requirement for cell growth and its breakdown during the culture. Moreover, nonessential amino acids have been added into a defined medium developed for hMSC growth [].A carbohydrate source is essential for the growth of cells in culture, since neither amino acids nor fats can readily be used either as the sole energy source or as substrates to build up a sufficient pool of intracellular carbohydrate intermediates. Glucose is the commonly provided source of energy for cells in culture, and together with amino acids it is included in most defined basal medium formulations. It is known that glucose at high concentrations has harmful effects on some cell types in culture. For this reason, low concentrations of glucose (~5 mM) are commonly used for hMSC culture. However, recent evidence reveals that high glucose concentrations (~25 mM) led to comparable or higher growth of animal and human MSCs [–].It is known that certain lipids, such as cholesterol, free linoleic acid and its metabolites, free oleic acid, and phospholipids containing linoleic acid, stimulate growth of mammalian cells. Lipids are frequently not included in the defined portions of cell culture media. Large amounts of bound lipids ar therefore, normally serum-containing media do not need the supplementation of additional lipids. In contrast, externally supplied lipids are generally required in serum-free culture []. Lipid supplements have been added to serum-free media for hMSCs [, ].In general, mammals require 12 vitamins, including the 4 fat-soluble vitamins (A, D, E, and K) and the 8 members of the B complex (thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, folacin, vitamin B12, and biotin). In addition, primates, guinea pigs and flying mammals require vitamin C (ascorbic acid). In contrast, normal diploid cells in culture exhibit requirements for the B vitamins, while some cells have shown growth responses to ascorbic acid. For this reason, most cell culture media include all the B vitamins but variably contain vitamin C []. Other vitamins are not included in media. The B vitamins function as cofactors for specific enzymes, and their deficiency can result in death of the animal. Ascorbic acid functions as an oxygen acceptor in several mixed-function oxidase systems. Rowe et al. reported that ascorbic acid had an effect on collagen synthesis by human diploid fibroblasts and played a role as a growth-promoting factor for many cell types []. The use of ascorbic acid for hMSC culture seems to be a matter of debate. Gronthos and Simmons demonstrated that ascorbic acid was a critical component under serum-free conditions for supporting the formation of CFU-F colonies in primary cultures of human BM cells []. We and others also demonstrated that ascorbic acid promoted hMSC growth [, ]. Moreover, it was observed that the lack of ascorbic acid in medium significantly reduced osteogenic potential of hMSCs []. On the other hand, ascorbic acid has typically been used as a supplement in some MSC differentiation media. Mimura et al. reported that ascorbic acid increased osteoblastic marker expression in hMSCs grown in a serum-free condition, and thus the authors removed this component from their defined medium formulation []. It should be noted that ascorbic acid is very labile under cell culture conditions []; therefore, care should be taken when this substance is used in cell culture.The salts that are included in most media are those of Na+, K+, Mg2+, Ca2+, Cl−,
, and they play several functions. The salts primarily contribute to retaining the osmotic balance of the cells. The osmolality of most cell culture media is approximately 300 mOsm/kg, and this represents an optimal value for most cell lines. It has been shown that many cell lines tolerate variation of approximately 10% of this optimal value, and thus care should be taken when extra salts are added into a medium []. Divalent cations, particularly Ca2+, are required by some cell adhesion molecules, such as the cadherins. Ca2+ also acts as an intermediary in signal transduction, and the concentration of Ca2+ in the medium can have an influence on cell proliferation or differentiation. Na+, K+, and Cl− regulate membrane potential, while
have roles as anions required by the matrix and nutritional precursors for macromolecules, as well as regulators of intracellular charge.
also plays a role as a buffer and its concentration is determined by the concentration of CO2 in the gas phase [].In addition to the inorganic salts, other inorganic elements, such as Mn2+, Cu2+, Zn2+, Mo6+, Va5+, Se8+, Fe2+, Ca2+, Mg2+, Si4+, Ni2+ are present in serum in trace amounts. These substances are referred to as trace elements and are included in most medium formulations. Although the role of these trace elements has been only partially elucidated, it has been demonstrated that many of these elements act as enzyme cofactors and are essential to the survival and growth of most cells [, ]. For instance, selenium is well recognized as an activator of glutathione peroxidase, a key enzyme essential for detoxifying cytotoxic oxygen radicals, and has been considered as an essential trace element for many cell types in culture [, , ]. It was also observed that selenium increased the proliferation of hMSCs from AT []. In contrast, numerous reports demonstrated that selenium suppressed cell proliferation in culture and induced cytotoxicity []. In our study, the addition of selenium into a serum-based medium reduced the colony-forming ability of hMSCs []. Considered together, the effect of selenium on hMSC expansion seems to depend on culture conditions or cell sources.4.1.2. Nonnutrient FactorsBeyond the nutrients, the growth of mammalian cells requires additional substances (provided from serum or other sources). These nonnutrient factors, including growth and attachment factors and hormones, generally function in regulatory roles on cellular differentiation as well as growth and proliferation. These regulatory factors are not included in most basal media and are frequently supplemented to serum-free media. Often, the requirements of nutrients for the growth of cells of interest could be satisfied by selecting appropriate therefore, identifying growth-stimulating regulatory factors has typically been the key subject in the development of serum-free media. Much effort has been made to investigate the effect of cytokines and growth factors on hMSC growth. Many reports showed that bFGF promotes the proliferation of hMSCs [, –]. TGF-β1 is also known to support hMSC proliferation in combination with other growth factors [, ]. It is important to note that TGF-β1 alone showed a growth-inhibitory effect on hMSCs, while demonstrating a significant degree of synergistic effect with bFGF []. The impact of PDGF on hMSCs is controversial. Several studies reported that PDGF enhanced the proliferation of MSCs from human and animal BM [, , ]. Moreover, PDGF-enriched plasma or platelet lysate has been shown to support the isolation and expansion of hMSCs. In contrast, our group observed that PDGF reduced considerably the colony-forming property of hMSCs []. It is presumed that the contradictory data are due, at least in part, to different modes of interactions with different factors present in different culture conditions. Other growth factors, such as EGF, Activin A, aFGF, and FGF4, have also been shown to promote hMSC proliferation (e.g., [, , ]); however, their effects could be masked in the presence of more potent factors (e.g., bFGF) [].Binding proteins such as albumin and transferrin are commonly added to serum-free media. Parker et al. reported that the absence of albumin in their serum-free formulation reduced hMSC growth []. Supplementation of key hormone components into serum-free media is also crucial. We observed no stimulatory effects of insulin and progesterone under FBS-based conditions in our study []. Nonetheless, these components showed growth-promoting effects for many cell types in serum-free condition []. Hydrocortisone has been shown to increase the proliferation of adherent human BM cells []. It has also been demonstrated that dexamethasone, a synthetic reagent of fluoridated hydrocortisone, is an essential component in serum-free medium for the growth of CFU-F colonies in primary cultures of human BM cells []. Interestingly, our experiments showed that the supplementation of hydrocortisone into an FBS-containing medium significantly inhibited cell proliferation and caused a change in cell morphology from spindle-shaped to cuboidal (data not shown); however, this hormone was found to be a key component in our serum-free media for hMSC growth, displaying a considerable combined effect with fetuin, particularly in primary culture []. This indicates that the effect of hydrocortisone is highly dependent upon culture conditions. Fetuin, a major plasma glycoprotein, has been used as a requirement for serum-free primary cultures of hMSCs and other cell types such as mouse fibroblast and epithelial cells [, ]. Heparin, a glycosaminoglycan that typically acts as an anticoagulant factor, has been shown to have proliferative or antiproliferative effects on various cell types [, ]. Addition of heparin into culture media led to a reduced growth of hMSCs from AT and BM [, ]. In contrast, Mimura et al. reported the growth-enhancing effect of heparin on a genetically modified hMSC line [].4.1.3. Other VariablesFor successful cell cultures, it is also important to control other key variables, particularly when serum-free media are used. Beyond the nutrients and nonnutri