中国间充质干细胞药物开发流程
isct_间充质干细胞_鉴定标准_理论说明
isct 间充质干细胞鉴定标准理论说明1. 引言1.1 概述在细胞治疗和再生医学领域，间充质干细胞（ISCT）作为一种重要的细胞资源，引起了广泛的关注。
ISCT具有多向分化潜能、免疫调节作用及促进组织修复的能力，被认为是一种理想的干细胞来源。
然而，由于缺乏统一和规范的鉴定标准，在实践应用中存在着不确定性和挑战。
1.2 文章结构本文将对ISCT进行深入探讨，并重点介绍其鉴定标准的理论说明。
文章包括以下几个部分：引言、ISCT间充质干细胞、鉴定标准理论说明、实践应用与挑战以及结论与展望。
1.3 目的本文旨在系统梳理当前关于ISCT鉴定标准的理论知识，并探讨该领域面临的实践应用和挑战。
通过对ISCT表面标记物使用、功能性特征评估方法以及分子生物学检测手段应用等方面原理的介绍，希望能够提供给读者一个全面的理论基础，并展望ISCT鉴定标准未来的发展方向。
同时，对于临床转化前景展望、存在的问题与挑战以及解决方案和研究进展等方面也将进行讨论。
以上是本文“1. 引言”部分的详细内容。
该部分概述了文章的主题和目的，并介绍了本文的结构。
接下来将继续展开描述ISCT间充质干细胞及其鉴定标准相关内容。
2. ISCT间充质干细胞2.1 定义与特征ISCT（国际干细胞治疗学会）定义了间充质干细胞（MSCs）作为一类多潜能的成体干细胞，具有自我更新和多向分化的能力。
这些细胞可以从不同来源获得，包括骨髓、脐带血、脂肪组织等。
ISCT认为，MSCs应满足以下三个基本标准：1) 在培养条件下，MSCs应具备黏附能力；2) MSCs应表达特定的表面标记物，如CD73、CD90和CD105，并且在同时应该缺乏（或仅有极低水平）CD34、CD45、HLA-DR等造血和免疫相关标记物；3) MSCs应能够分化为成骨细胞、软骨细胞和脂肪细胞等不同种类的细胞。
此外，ISCT提出了额外的功能性特点来进一步确认MSCs。
这些功能性特点包括：1) 免疫调节作用：MSCs可以抑制免疫反应，并通过调节T淋巴细胞、B 淋巴细胞和自然杀伤细胞等免疫细胞的功能来达到免疫调节作用；2) 细胞迁移能力：MSCs具有从注射部位向受损组织迁移的能力；3) 分泌多种生物活性分子：MSCs可以产生多种生长因子、细胞因子和表观遗传调控因子，对损伤修复和组织再生具有重要作用。
中国制药企业小分子创新药物研发流程
中国制药企业小分子创新药物研发流程中国制药企业小分子创新药物研发流程如下：1、药物靶点发现及确认：这是新药研发的第一步，涉及从有效单体化合物着手发现药物靶点；通过定量分析和比较研究在正常和疾病状态下蛋白质表达谱的改变发现靶点；以正常组织与病理组织基因表达差异发现靶点；以蛋白质相互作用为基础发现药物靶点；应用RNA干扰技术特异的抑制细胞中不同基因表达，通过细胞的表型变化发现靶点等。
2、苗头化合物的筛选：靶点确认后，需要对其作用靶点进行确认和验证，当药物的作用靶点得到验证和确认之后，研究工作者就会根据靶点的空间结构设计一系列可与其匹配的分子结构，这些被称为苗头化合物（Hit）。
3、苗头化合物的验证和优化：这一阶段主要对苗头化合物进行体外和初步体内验证，评估其药理、毒理作用机制和体内外药代动力学特征，确定先导化合物。
4、临床前研究：这一阶段包括药效学研究、药代动力学研究和毒理学研究。
药效学研究主要考察药物对实验动物模型的治疗作用及作用机制；药代动力学研究主要是对动物体内的药物浓度进行检测，评估药物的吸收、分布、代谢和排泄情况；毒理学研究包括单次给药毒性试验、重复给药毒性试验、生殖毒性试验、致突变与致癌毒性试验以及依赖性试验等。
5、新药申报：完成临床前研究后，企业需要向国家药品监督管理部门申报临床试验。
6、临床试验：临床试验分为Ⅰ期、Ⅱ期、Ⅲ期和Ⅳ期。
Ⅰ期临床试验主要考察药物的安全性和耐受性；Ⅱ期临床试验主要评估药物的疗效和安全性；Ⅲ期临床试验旨在进一步验证药物的疗效和安全性，并考察其在更大患者群体中的效果；Ⅳ期临床试验是在新药上市后的观察性研究阶段，评估药物在实际使用中的疗效和安全性。
7、新药上市申请：完成临床试验后，企业需要向国家药品监督管理部门提交新药上市申请。
8、新药上市后监测：新药上市后，企业需要对新药的疗效和安全性进行持续监测，定期向国家药品监督管理部门报告相关数据。
实际流程可能根据具体情况有所不同。
含间充质干细胞的注射用玻璃酸钠
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安全性高：经过严格的临床试 验和安全性评估，证明本品的 安全性较高，不良反应较少。
疗效显著：对骨关节炎等疾病 具有显著的治疗效果，能明显 改善患者的疼痛和活动能力。
使用方便：本品为注射剂型， 使用方便，可由医生在诊所或
医院进行注射治疗。
产品适用范围与剂量
适用范围
本品适用于骨关节炎、半月板损伤、 关节软骨损伤等疾病的辅助治疗。
新产品开发
随着对间充质干细胞和玻璃酸钠的深入研究，将会有更多新 型的、更加有效的含间充质干细胞的注射用玻璃酸钠产品被 开发出来。这些新产品将进一步改善患者的治疗效果和生活 质量。
市场需求增长趋势及影响因素分析
市场需求增长
随着人们对生物医学技术的认识不断提高，对含间充质干细胞的注射用玻璃酸钠的市场需求也将不断 增长。特别是在骨关节炎、腰椎间盘突出、眼科疾病等领域的广泛应用，将进一步推动市场需求的增 长。
剂量
具体剂量应由医生根据患者的病情和 医生的建议来确定，一般每次注射量 为1-2毫升。
制作工艺与质量控
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制作工艺流程
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细胞来源
获取并筛选合适的间充质 干细胞来源，如脐带、胎 盘等。
细胞培养
在无菌条件下，将筛选出增
通过细胞分裂增殖，使细 胞数量增加。
生产效率低下
由于生产过程复杂，可能存在生 产效率低下的问题，影响产品的
上市时间和市场供应。
对策建议及防范措施
加强生产环境的监控和检查
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通过加强生产环境的监控和检查，确保生产环境的卫生和安全
，从而降低产品污染的风险。
优化生产工艺和流程
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通过优化生产工艺和流程，减少批次间的差异，提高产品的稳
干货间充质干细胞功能及应用
路漫漫其修远兮, 吾将上下而求索
Annu Rev Pathol 2011;6457-478
无处不在
牙髓
外周血
脐带
MSC
胸腺 羊膜
路漫漫其修远兮, 吾将上下而求索
骨髓
脂肪
脐带血
富含MSC的人体组织
路漫漫其修远兮, 吾将上下而求索
图片来自网络
年龄与骨髓MSC含量的变化
路漫漫其修远兮, 吾将上下而求索
路漫漫其修远兮, 吾将上下而求索
稳定性评价
P30
Although hUC-MSCs with genomic alterations do not undergo malignant transformation, periodic genomic monitoring and donor management focusing on genomic stability are recommended before these cells are used for clinical applications. (Cell Death Dis 2013;4e950)
稳定性评价
微生物
• 病毒 • 细菌 • 支原体
辅料素
• 内毒素 • 胎牛血清残留
生产工艺
稳定性评价
P14
P18 Array-CGH and cytogenetic analyses showed that BM-MSCs expanded in vitro did not show chromosomal abnormalities. Telomerase activity and hTERT transcripts were not expressed in any of the examined cultures and telomeres shortened during the culture period. (Cancer Res 2007;67(19):9142-9149)
脐带间充质干细胞PPT
分离与培养技术
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酶消化法
使用特定的酶如胶原酶、 胰酶等分解脐带组织，释 放出间充质干细胞。
组织块贴壁法
将脐带组织切割成小块， 贴壁培养，让间充质干细 胞从组织块中爬出并扩增。
流式细胞术分离法
利用间充质干细胞的表面 标记物，通过特定的抗体 进行免疫分离。
诱导分化与基因编辑
再生医学
组织工程
脐带间充质干细胞具有多向分化潜能，可诱导分化为多种组 织细胞，如骨细胞、软骨细胞、脂肪细胞等。在组织工程领 域，可用于构建人工组织器官，为器官移植提供新的来源。
损伤修复与再生
脐带间充质干细胞可分泌多种生长因子和细胞因子，具有促 进损伤组织修复和再生的能力。在损伤修复领域，可用于治 疗烧伤、创伤等，促进受损组织的再生与修复。
技术挑战与解决方案
技术挑战
脐带间充质干细胞的分离、培养和分化等技术在实践中仍面临一些技术难题，如低分离效率、低细胞活率等。
解决方案
加强基础研究，深入了解脐带间充质干细胞的生物学特性和调控机制；采用先进的分离和培养技术，提高细胞的 分离效率和活率；加强国际合作，共享技术和资源，推动干细胞技术的进步。
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脐带间充质干细胞的挑战与解 决方案
伦理挑战与解决方案
伦理挑战
脐带间充质干细胞的研究和应用涉及到伦理问题，如胚胎和胎儿的权益、人类基因编辑 等。
解决方案
建立严格的伦理审查机制，确保研究符合伦理标准；加强公众教育和科普，提高公众对 干细胞研究的认知和理解；尊重和保护受试者的权益，确保研究过程的公正和透明。
社会影响与普及程度
疾病治疗
脐带间充质干细胞在多种疾病的 治疗中展现出巨大潜力，有望为
2015-新版-GMP标准制备间充质干细胞
Standard Operating Procedure for the Good Manufacturing Practice-Compliant Production of Human Bone MarrowMesenchymal Stem CellsLivia Roseti,Marta Serra,and Alessandra Bassi AbstractAccording to the European Regulation (EC 1394/2007),Mesenchymal Stem Cells expanded in culture forclinical use are considered as Advanced Therapy Medicinal Products.As a consequence,they must be produced in compliance with Good Manufacturing Practice in order to ensure safety,reproducibility,and efficacy.Here,we report a Standard Operating Procedure describing the Good Manufacturing Practice-compliant production of Bone Marrow-derived Mesenchymal Stem Cells suitable for autologous implan-tation in humans.This procedure can be considered as a template for the development of investigational medicinal Mesenchymal Stem Cells-based product protocols to be enclosed in the dossier required for a clinical trial approval.Possible clinical applications concern local uses in the regeneration of bone tissue in nonunion fractures or in orthopedic and maxillofacial diseases characterized by a bone loss.Keywords:Bone marrow,Mesenchymal stem cells,Good manufacturing practice,Quality control,Advanced therapy medicinal products,Bone regeneration1IntroductionMesenchymal Stem Cells (MSCs)display a low density in bonemarrow,thus an in vitro expansion step is required in order to obtain a cell number suitable for clinical applications (1–3).Euro-pean regulations define such expanded cells as Advanced Therapy Medicinal Products (ATMPs)that must be produced in accordance with the current Good Manufacturing Practice (GMP)(4,5).Those rules,which have been already encoded for conventional drugs,allow for the manufacture of medicinal products in a stan-dardized and controlled way minimizing,at the same time,con-tamination risks.Here,we report a Standard Operating Procedure (SOP)describing the multistep GMP-compliant manual production of Bone Marrow-derived MSCs to be used for autologous implanta-tion in humans (6–8).MSCs,isolated from an eparinated (1,000I.U.of heparin)bone marrow sample harvested from the iliac crest of adult donors (9,10)(about 10–20ml),are expanded in monolayerMethods in Molecular Biology (2015)1283:171–186DOI 10.1007/7651_2014_103©Springer Science+Business Media New York 2014Published online:05August 2014w w w wc el l 8.c n w w w .s t e mc e l l 8.c n w w w .s t em c c e l l 8.c n ww w .s t e m c e l l 8.c n w ww.s t e mc c el l 8.c n w w w.s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc cel l 8.c n w .s t e mc e l l 8.c n w .s t em cup to 3weeks.The final product is a small volume MSC suspension suitable for the clinical use only after passing proper quality con-trols.In fact,the procedure involves several GMP’s mandatory samplings for environmental and cell quality control analyses (3,11)that must be carried out by specialized laboratories and will not be described in detail.Previous validation studies have stated final product’s shelf-life (properly named stability):we demonstrated that MSC’s features (sterility,viability,and phenotype)are maintained at acceptable levels when the MSC suspension is stored at room temperature for a maximum of 72h.After this time the product expires and can no longer be transplanted.To reach full GMP-compliance,the process generates two types of reference samples collected from the final product—cells and supernatant—which should be both stored for at least 1year after implantation and analyzed if issues arise (12).If implantation is delayed for organizational reasons or patient’s illness or if repeated doses at different times are required,cells are stored in liquid nitrogen as intermediate product.This SOP can be utilized as a template for the development of investigational medicinal Mesenchymal Stem Cells-based product protocols to be enclosed in the dossier required for a clinical trial approval.Possible clinical applications concern local uses (cells alone or in combination with biomaterials and/or growth factors)for the regeneration of bone traumatic or degenerative damages,pseudoarthrosis and defects of consolidation,congenital disorders or maxillofacial injuries (13–15).2Materials2.1Raw MaterialsCell culture reagents suitable for cell therapy applications should bepurchased from Companies that guarantee their GMP compliance production.Sterility should be certified by specific analyses,pre-formed in compliance with the requirements of the European Pharmacopoeia,current edition (16).All reagents used in this protocols are:Dulbecco’s Modified Eagle Medium (DMEM)low glucose (1g/l)(basal medium);Dul-becco’s Phosphate Buffered Saline (PBS)(1Â);Fetal Bovine Serum (FBS)Pharma Grade,Australian Origin;L -Glutamine 200mM (recombinant origin)and Trypsin-Ethylenediaminetetraacetic acid (EDTA)(1:250)1Â(porcine source).Trypsin-EDTA and FBS must be also certified to be free from porcine and bovine mycoplasmas and viruses,respectively.In addi-tion,FBS must be attested to be produced in a Bovine Spongiform Encephalopathy/Transmissible Spongiform Encephalopathy172Livia Roseti et al.c el l 8.c n w ww .s t e mc e l l 8.c n www .s t em c cel l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc c el l 8.c n w w w.s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc cel l 8.c n w .s t e mc e l l 8.c n w .s t em c(BSE/TSE)free Country,such as Australia or New Zealand,andscreened for prion absence (17).L -Glutamine’s recombinant origin must be clearly indicated and certified too.CryoSure-Dimethyl Sulfoxide (DMSO)is purchased from WAK–Chemie Medical GmbH,Germany.Plastic material must be certified to be sterile,pyrogen free (latex free whenever necessary)and tested for cell culture (or stem cell tested).Vented culture flasks are recommended.To manage high cell numbers we use a multilayer flasks system (Hyperflask vessels,Corning Life Sciences).Double or triple packages should be preferred to eliminate/reduce the need for alcohol wipe downs and similar procedures required in clean room production facilities.Quality control sampling:–BacT/ALERT 1Aerobic Culture Bottles (SA)(BioMerieuxInc.,Marcy L’Etoile,Craponne,France)(18).–BacT/ALERT 1Anaerobic Culture Bottles (SN)(BioMerieuxInc.,(Biomerieux Industry,Marcy L’Etoile,Craponne,France)(18).–Apirogenic Bottles for endotoxin testing (Charles River Labor-atoires,l’Arbresle,France).–PCR tubes for Real time PCR detection of Mycoplasma contam-ination (Venor 1GeM-qDual Mycoplasma Detection Kit for Real-Time PCR,Minerva Biolabs GmbH,Berlin,Germany).–Settle and contact plates for environmental microbiological con-trol and glove prints (Heipha Dr M €uller GmbH,Eppelnheim,Germany).–Particle counter for environmental particle control.2.2LocationIn Europe,a GMP-facility (Cell Factory)allowed to ATMP pro-duction by the competent authority is required (5).A GMP-facilityconsists in environments where specific parameters such as air filtration and ventilation,temperature,relative humidity,differen-tial pressure,number of air particles,and bacterial colony forming units are standardized and continuously monitored.The structure includes clean rooms of different classification up to A (the local zone for high-risk operations i.e.biohazard hood)in B work places (the background environment for the grade A zone),according to European current GMP (5).High-risk operations include manip-ulations where the cells are exposed to environment such as trypsi-nization and medium change.For lower-risk operations,such as medium warming or centrifugation,grade B areas are appropriate.Only trained and equipped personnel are admitted to the facility.GMP Production of Mesenchymal Stem Cells173c el l 8.c n w ww .s t e mc e l l 8.c n www .s t em c cel l 8.c n w w w.s t e mc e l l 8.c n w ww.s t e mc ce l l 8.c n w ww.s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc cel l 8.c n w .s t e mc e l l 8.c n w .s t em cOperator’s equipment includes protective and disposable clothes towear(Fig.1).Raw materials must be introduced separately frompersonnel,through a clean pass box.2.3Equipment Biohazard hood;CO2incubator(set at37 C temperature,5%CO2and95%relative humidity);centrifuge;phase contrast micro-scope;incubator(work chamber temperature of37 C);vortex;chilling plate/refrigerated racks;cell counter;refrigerator/freezer;liquid nitrogen tank.3Method3.1Cell Isolation and Seeding (See Notes1–4)1.Warm/thaw basal medium,L-glutamine and FBS in theincubator.2.Under the biohazard hood prepare complete medium(basalmedium supplemented with10%FBS and4mM L-glutamine): discard by aspiration60ml from a500ml bottle of basal medium,add10ml L-glutamine and50ml FBS;resuspend with a pipette.Fig.1Disposable sterile garments for cell manipulation.Cell manipulating operators as well as personnel must wear disposable garments that ensure biological products protection.Only trained and equipped operators can enter the GMP-facility174Livia Roseti et al.c e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw.s t e mc e l l8.c nw.st e mc3.Aspirate the eparinated bone marrow with a 18-G needle inserted in a syringe and transfer in sterile tube/s.4.Resuspend and measure bone marrow amount with a pipette.5.Dilute the bone marrow samples 1:4with complete mediumand resuspend.6.Harvest samples for microbiological control:–1ml with a sterile syringe to inoculate in a BacT/ALERT 1Aerobic Culture Bottle (SA)(Fig.2);–1ml with a sterile syringe to inoculate in a BacT/ALERT 1Anaerobic Culture Bottle (SN)(Fig.2);–2ml to collect in PCR tube for mycoplasma detection.Collected samples are then to be sent to the proper specializedquality control laboratories that will perform the analyses (see Note 5).7.Seed cells in culture flasks,typically T75(7ml/T25;14ml/T75;28ml/T150;560ml/multilayer flasks).8.Place the flasks in the CO 2incubator.9.Store the complete medium at 4 C.Fig.2Quality control sampling on Mesenchymal Stem Cells.To harvestsamples for microbiological control during critical steps,1ml of supernatant is inoculated into a BacT/ALERT ®Aerobic Culture Bottle (SA)and 1in a BacT/ALERT ®Anaerobic Culture Bottle (SN)with a sterile syringe (see Note 5).The bottles are then sent to a GMP-compliant Quality Control Laboratory for the analysisGMP Production of Mesenchymal Stem Cells175c el l 8.c n w ww .s t e mc e l l 8.c n w ww .s t em c cel l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc c el l 8.c n w w w.s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w w w.s t e mc e l l 8.c n w ww.s t e mc ce l l 8.c n w .s t e mc e l l 8.c n w .s t em c3.2First Medium Change(See Notes 1and2)1.After3days,warm complete medium and PBS in the incubator.2.Pull out theflasks from the CO2incubator and observe themacroscopic appearance of the culture medium:–if the medium is opaque or dense and yellow there is the suspect of a contamination.In this case the procedure isstopped and sampling for microbiological control isneeded(see Section3.1);–if the appearance is clear and the color red or dark red go on with this procedure.3.Under the biohazard hoodfilter the amount of completemedium required for processing with a vacuumfiltration sys-tems(or with afilter and a syringe).4.Aspirate medium from theflasks and place it in sterile tubes.5.Centrifuge the medium at500Âg for7min,at roomtemperature.6.In the meantime add to theflasks the PBS solution(5ml forT25flasks,10ml for T75flasks,20ml for T150flasks,100ml for multilayerflasks)in order to wash out stromal matrix debris and red blood cells(see Note6and Fig.3a).7.Close theflasks and placed them horizontally for1min,gentlyshaking.8.Aspirate and discard the PBS.9.Run a second wash(only steps7and8).10.Evaluate the cultures under a phase-contrast microscope(Fig.3b),noting the following parameters:cell adhesion (absent,present,partial,poor);cell density(confluence,non-confluence,next to the confluence);cells in the supernatant (presence,absence);matrix/red blood cells(presence, absence);unusual morphology.11.Transfer theflasks under the biohazard hood and discardthe PBS.12.Transfer the centrifuged tubes under the biohazard hood,aspirate half supernatant volume of that normally used to eachflask(3.5ml for the T25flasks,7ml for T75flasks, 14ml for T150flasks,280ml for multilayerflasks)(see Note6).13.Add to theflasks the same amount of complete fresh mediumand resuspend.14.Place theflasks in the CO2incubator.15.Store reagents at4 C.176Livia Roseti et al.c e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw.s t e mc e l l8.c nw.st e mc3.3Trypsinizationand Intermediate Product Freezing(See Notes 1,2,and 7)1.After 3–4days,macroscopically evaluate culture medium,not-ing the following parameters:clarity/turbidity,color (red orange,red violet,yellow).2.Check the cells under a phase-contrast microscope noting thefollowing parameters:cell adhesion (present,absent,poor);confluence (70–80%confluent,nonconfluent,next to the confluence);cell presence or absence in the supernatant;matrix/red blood cells (presence,absence);unusual morphology.3.Make a decision based on both observations (see steps 4,5,6,and 7).Fig.3Phase-contrast microscope morphological observation of Mesenchymal Stem Cells at different steps of the Standard Operating Procedure.(a )Three days after seeding (First medium change )red blood cells and matrix hamper observation.(b)Adherent cells become more visible only after two PBS washes .Matrix debris and/or red blood cells are still evident and should disappear in a few days.(c)Cells have reached 70–80%confluence and trypsinization is recommended.(d)Full cell detachment occurs as a suspension of cells with round morphology after Trypsin-EDTA incubation.All images were taken with a digital camera (magnification Â4)GMP Production of Mesenchymal Stem Cells177c el l 8.c n w ww .s t e mc e l l 8.c n w ww .s t em c cel l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc c el l 8.c n w w w .s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc cel l 8.c n w .s t e mc e l l 8.c n w .s t em c4.If 70–80%confluence is not reached,trypsinization should be delayed until reached and a medium change is needed.5.In particular,change the culture medium with complete medium in the case of red medium color and/or adhesion;change the culture medium with a 20%FBS supplement in the case of violet medium color and/or poor adhesion:–warm complete medium and FBS (if necessary);–prepare 10or 20%FBS medium;–transfer the cultures under the biohazard hood;–filter the needed amount of medium,depending on flasks size and number;–aspirate the medium from the flasks and add propermedium—complete or with 20%FBS—(7ml for T25flasks,14ml for T75flasks,28ml for T150flasks;560ml for multilayer flasks);–close and place flasks in the CO 2incubator.6.Trypsinization and freezing can be performed in case of about70–80%confluence (Fig.3c ):–prepare complete medium if necessary;–warm complete medium and PBS in the incubator;–thaw Trypsin-EDTA in the incubator;–filter the amount of reagents required for processing;–transfer the cultures under the biohazard hood;–aspirate and discard the supernatants;–add the PBS wash solution (5ml for T25flasks,10ml for T75flasks,20ml for T150flasks,and 100ml for multilayer flasks);–close and place horizontally the flasks,gently shaking for 30–60s;–run a second wash;–add the Trypsin-EDTA solution (4ml for T25flasks,7ml for T75flasks,14ml for T150flasks,and 100ml for multilayer flasks);–close and place the flasks in the CO 2incubator for 15min toallow cell detachment;–pick the flasks and check the cells under the phase-contrast microscope to verify cell detachment (which occurs as a suspension of cells with round morphology)(Fig.3d );–if some cells are still adherent to the substrate,put the flasksagain in the CO 2incubator for 3–5min to allow full detachment;178Livia Roseti et al.c el l 8.c n w w w .s t e mc e l l 8.c n www .s t em c ce l l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc c el l 8.c n w w w.s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc cel l 8.c n w .s t e mc e l l 8.c n w .s t em c–if detachment is completed,inactivate the Trypsin-EDTA solution by adding complete medium to theflasks under the biohazard hood(8ml for T25flasks,14ml for T75flasks, 28ml for T150flasks,and200ml for multilayerflasks);–resuspend and transfer to50ml tubes;–collect a0.5ml sample for cell counting;–centrifuge7min at500Âg,at room temperature;–in the meantime count the cells;–under the biohazard hood open the centrifuged tubes,care-fully discard the supernatants and gently shake the pellets (see Note8);–set the number of cryovials to use for freezing:1cryovial up to3Â106Æ0.5Â106MSCs;–place the empty cryovials in a refrigerated rack;–prepare the cell freezing mixture in a tube:0.9ml of com-plete medium,0.2ml of DMSO and0.9ml of FBS(mixture for one cryovial)(see Note7);–resuspend,filter,and place the mixture in the refrigerated rack;–carefully discard the supernatant of the centrifuged tubes and gently resuspend the cell pellet(see Note8);–add1.6ml of the freezing mix to each pellet,drop by drop, using a sterile pipette while gently shaking the tubes with the other hand;–gently resuspend and transfer into the cryovials;–close the cryovials and house them in a storage box that enables a slow decrease of the temperature(about1 C per minute);–exit the cleanroom and place the container into a freezer set at a75 C temperature;–after about24h place the cryovials in the vapor phase of a liquid nitrogen tank(see Note7).7.Collect samples for microbiological tests in case of suspectedcontamination(yellow color,unusual morphology)as in Section3.1and stop the procedure until results(see Note5).3.4Intermediate Product Thawing and Seeding(See Notes1, 2,5,and7)1.Open the liquid nitrogen tank and harvest the cryovials,check-ing their integrity and identity.2.Get samples in the cleanroom(through the pass box)within arefrigerated rack.3.Prepare and warm a20%FBS supplemented medium.4.Place the cryovials in the incubator.GMP Production of Mesenchymal Stem Cells179c e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw.s t e mc e l l8.c nw.st e mc5.After3–4min check if the cell suspension appears thawed.6.If small ice chunks are still evident put the samples again in theincubator for30–60s.7.When no more ice residues are visible,place the cryovials underthe biohazard hood and transfer each cell suspension in a tube containing18ml of the20%FBS medium.8.Close the tubes and vortex the solution for about10s.9.Centrifuge at500Âg for7min,at room temperature.10.Transfer the supernatant into sterile tubes for quality controlsampling as in Section3.1.11.Gently shake the pellets,add20%FBS medium(7ml for T25flasks,14ml for T75flasks,28ml for T150flasks,560ml for multilayerflasks)and resuspend(see Note8).12.Seed at low density in an adequate number of cultureflasks(seeNote9).13.Check cell presence under the phase-contrast microscope.14.Place theflasks in the CO2incubator.3.5Expansion(See Notes1,2,and10)1.Pull outflasks from the CO2incubator.2.Macroscopically evaluate culture medium,noting the followingparameters:clarity/turbidity,color(red orange,red violet, yellow).3.Check the cells under a phase-contrast microscope noting thefollowing parameters:cell adhesion(present,absent,poor);confluence(confluent,nonconfluent,next to the confluence);cell presence or absence in the supernatant;matrix/red blood cells(presence,absence);unusual morphology.4.Make a decision based on both observations as detailed below.5.Change the culture medium with complete medium in the caseof red medium color and/or cell adhesion;change the culture medium with a20%FBS supplement in the case of violet medium color and/or poor adhesion,as described in Section3.3.6.Trypsinization in case of about70–80%confluence(seeNote10)(Fig.3c)or if matrix debris and/or red blood cells are still evident(see Note6)(Fig.3a,b):–trypsinize(until centrifugation step)and count the cells,as detailed in Section3.3–add to the pellets the necessary amount of complete medium in order to allow a low density seeding,resuspend and place in cultureflasks(14ml for T75flasks,28ml for T150flasks,560for the multilayerflasks)(see Note9);180Livia Roseti et al.c e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw.s t e mc e l l8.c nw.st e mc–check cell presence in theflasks under the phase-contrast microscope;–place theflasks in the CO2incubator.7.Collect samples for microbiological tests in case of suspectedcontamination(yellow color,unusual morphology)as in Sec-tion3.1and stop the procedure until results(see Note5).3.6Final Product Packaging and Reference Samples Generation(See Notes 1and2)1.Warm reagents and preparefinal medium:basal medium sup-plemented with L-glutamine(no FBS)(see Note11).2.Trypsinize and count the cells as detailed in Section3.3,untilcentrifugation step.3.Gently shake the pellets and add40mfinal medium(see Note8).4.Centrifuge7min at500Âg,at room temperature.5.Repeat steps3and4(see Note11).6.Under the biohazard hood transfer the supernatant into steriletubes from which to collect the following samples that are then to be sent to the proper specialized laboratories of quality control:–1ml with a sterile syringe to inoculate in a BacT/ALERT1 Aerobic Culture Bottle(SA);–1ml with a sterile syringe to inoculate in a BacT/ALERT1 Anaerobic Culture Bottle(SN)(see Note5);–2ml with a sterile pipette to be placed in a proper tube for PCR mycoplasma detection;–0.5ml with a sterile pipette to inoculate in an a-pyrogenic Bottle for endotoxin testing;–0.5ml with a sterile pipette to be placed in a tube and frozen as counter sample atÀ80 C.7.Gently shake the pellets,addfinal medium in order to reach a1–2Â106cell concentration.Resuspend and collect the following samples which are then to be sent to the proper specialized laboratories of quality control (see Notes5and12):– 1.2Â106cells in micro-tubes for the phenotypical analysis (immuno-phenotype and tri-lineage ability);– 2.0Â106cells for karyotyping;– 1.0Â106cells in a tube as a counter sample to be frozen as in Section3.3.8.Transfer cell suspension into proper,identified tubes(primarycontainer)(Fig.4a)GMP Production of Mesenchymal Stem Cells181c e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw.s t e mc e l l8.c nw.st e mc9.Close each tube and put it in a secondary plastic sterile bag(secondary container)(Fig.4a ).10.Close and insert in outer jar container (Fig.4b ).11.Put a label on the outer container,which must contain(Fig.4b ):–identification of the Manufacturer;–identification of the product;–indication of the use (i.e.autologous use only);–batch number;–identification code of the patient;–expiration date;–signature of the person in charge.12.Exit the Cleanroom and store the final product at room tem-perature for a maximum of 72h.13.For the shipment use a container with lock (Fig.4c ).4Notes1.To avoid the risk of cross-contamination during manipulation it is required to use disposable materials and not to share reagents between cultures.Moreover,it is not possible to simultaneously process cultures from different patient,but only sequentially and after decontamination of the biohazard hood and relative equipment.2.Sampling for environmental (microbiological and particles)control is required during each phase of the process.At theFig.4Final product packaging.The final cellular products is transferred into proper tubes (primary container,a ),then put it into secondary sterile bags (secondary container,a ).The product is then inserted in a plastic jar,properly labeled (a andb ).For the shipment a container with lock is utilized (c )182Livia Roseti et al.c el l 8.c n w ww .s t e mc e l l 8.c n www .s t em c cel l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc c el l 8.c n w w w .s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc cel l 8.c n w .s t e mc e l l 8.c n w .s t em cend of cell manipulation,each operator should take glove printson agar plates.Recommended limits for contamination are indicated in EudraLex-Volume 4GMP Guidelines (5).3.A peripheral blood sample from each patient is needed in order to evaluate the presence of transmissible pathologies (Acquired Immune Deficiency Syndrome,Hepatitis B and C,and syphilis)(12).If a GMP-facility does not have the possibility to manipu-late the infected cells in separated areas,positive patients must be excluded.Therefore,manipulation can start only after nega-tivity is stated.This usually takes 1or 2days.In the meantime the bone marrow samples can be stored at 5Æ3 C.4.Density-gradient separation method is considered a standardstep for MSC isolation from bone marrow.However,such a procedure is time consuming and reagents are not often approved for clinical use.Therefore many GMP-facilities are trying to develop alternative methods,such as direct or diluted bone marrow seeding (9).5.Microbiological control and Mycoplasma analysis are per-formed in the critical steps of isolation,thawing,and final product packaging.Isolation and thawing criticism have been established by our GMP-facility on the basis that cell material is newly introduced or reintroduced in the cleanroom,respec-tively.Being “final product packaging”the most critical step since cells are then implanted in the patient,additional sam-pling for quality control analyses are required,such as for endotoxin detection,viability,phenotype,and genotype stabil-ity evaluation (4,5,7).If a cell contamination is detected,the process must be stopped.Since the here described orthopedic applications are not lifesaving,contaminated cells cannot be sterilized by irradiation or other treatments and must be discarded.6.In the “first medium change step”matrix and red blood cellspresence (Fig.3a )should be reduced by two PBS washes.This makes also possible to check cell presence under the phase-contrast microscope (Fig.3b ).Usually,debris gradually reduces until disappearance in 7-8days.However,if residues still persist in the cultures,a trypsinization,which in general dissolves all of them,is recommended.The first medium change is then per-formed using half of the centrifuged supernatants of the isola-tion step and half fresh complete medium.The supernatants,centrifuged in order to remove matrix debris and red blood cells,are utilized since rich of soluble factor produced by the cells themselves that can be useful for adhesion and growth.On the other hand,since it can carry also catabolic factors,fresh medium is added to enhance cell metabolism.GMP Production of Mesenchymal Stem Cells183c el l 8.c n w ww .s t e mc e l l 8.c n www .s t em c cel l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc c el l 8.c n w w w.s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc cel l 8.c n w .s t e mc e l l 8.c n w .s t em c。
间充质干细胞产品及其外泌体在炎症性肠病治疗中的研究进展
间充质干细胞产品及其外泌体在炎症性肠病治疗中的研究进展杨婧雯1，陈 芊1，单云龙1，刘嘉莉1，尉 宁1,2，王 婧2，王广基1*，周 芳1**（1中国药科大学药物代谢动力学重点实验室, 南京 210009；2江苏睿源生物技术有限公司, 南京211103）摘 要 炎症性肠病（inflammatory bowel disease, IBD ）发病机制不明，特征为进行性和终身复发性消化道炎症反应。
尽管现阶段新的治疗药物和策略不断涌现，但治疗作用局限于单一的抗炎功能，在复杂黏膜免疫环境下易出现耐药导致治疗失败。
间充质干细胞（mesenchymal stem cells, MSCs ）能定向归巢到结肠炎症部位，具有强大的免疫调节能力，可重塑肠道免疫环境和修复上皮屏障，为药物难治性患者的治疗提供了极具潜力的替代方案。
本文对MSCs 产品及其衍生的外泌体在临床上的应用、作用机制和工程化进行综述，以期为MSCs 及其外泌体产品用于IBD 的治疗提供参考。
关键词 炎症性肠病；间充质干细胞；外泌体；工程化中图分类号 R574 文献标志码 A文章编号 1000−5048(2024)01−0103−12doi ：10.11665/j.issn.1000−5048.2023113001引用本文 杨婧雯，陈芊，单云龙，等. 间充质干细胞产品及其外泌体在炎症性肠病治疗中的研究进展[J]. 中国药科大学学报，2024，55（1）：103 − 114.Cite this article as: YANG Jingwen, CHEN Qian, SHAN Yunlong, et al . Research progress on mesenchymal stem cell products and their exosomes in the treatment of inflammatory bowel disease[J]. J China Pharm Univ , 2024, 55(1): 103 − 114.Research progress on mesenchymal stem cell products and their exosomes in the treatment of inflammatory bowel diseaseYANG Jingwen 1, CHEN Qian 1, SHAN Yunlong 1, LIU Jiali 1, WEI Ning 1,2, WANG Jing 2, WANG Guangji 1*,ZHOU Fang 1**1Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009;2Renocell Biotechnology Co., Ltd., Nanjing 211103, ChinaAbstract Inflammatory bowel disease (IBD), whose pathogenesis remains elusive, is a group of autoimmune diseases characterized by chronic, progressive, and lifelong inflammation of the digestive tract. The pathogenesis of IBD remains elusive. Although a number of drugs have been developed to treat IBD, their effects are merely anti-inflammatory. In addition, current treatments for IBD are easily susceptible to resistance in clinical practice.Mesenchymal stem cells (MSCs) have been reported to have the ability to migrate to the site of inflammation,with potent immunoregulatory effects, and to rebalance the immune microenvironment and restore the integrity of the epithelial barrier with significant value of application, particularly for patients who are refractory to classic medicines. In this paper, we reviewed the clinical applications, mechanisms and engineerable properties of MSC products and their exosomes to provide some reference for the use of MSCs and their exosomes in the treatment of IBD.Key words inflammatory bowel disease; mesenchymal stem cells; exosomes; engineering收稿日期 2023-11-30 通信作者 *Tel ：************ E-mail ：*************************Tel ：************ E-mail ：**************基金项目 国家自然科学基金项目（No. 82073928）；南京市生命健康科技专项（No.202110006）；细胞生态海河实验室创新基金项目（22HHXBSS00005）；江苏省南京市联合资助项目（SBK2023070039）学报 2024, 55(1): 103 − 114103This study was supported by the National Natural Science Foundation of China (No. 82073928); the Nanjing Life and Health Science and Technology Program (No.202110006); the Haihe Laboratory of Cell Ecosystem Innovation Fund (22HHXBSS00005); and the Co-funded Programs in Nanjing, Jiangsu Province (SBK2023070039)炎症性肠病（inflammatory bowel disease, IBD）包括溃疡性结肠炎（ulcerative colitis, UC）和克罗恩病（Crohn's disease, CD），是由环境、免疫系统、肠道微生物组和个体遗传等因素的复杂互作引起的一类自身免疫性疾病，其一线治疗药物包括糖皮质激素、免疫抑制剂、抗生素和抗肿瘤坏死因子-α（tumor necrosis factor, TNF-α）疗法。
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我国间充质干细胞药物开发流程
随着生物医药科技的不断发展，干细胞疗法作为一种前沿的治疗方式，备受关注。
间充质干细胞作为一种重要类型的干细胞，具有多向分化
潜能和免疫调节作用，被广泛应用于临床疾病治疗。
本文将着重介绍
我国间充质干细胞药物开发的流程，以便让读者对该领域有一个全面
的了解。
1. 药物研发立项
间充质干细胞药物开发的第一步是药物研发立项。
这一阶段涉及确定
研发目标、搭建研发团队、制定开发计划和预算等工作。
需要进行市
场调研和竞品分析，明确产品的市场前景和竞争优势，为后续的研发
工作提供依据。
2. 干细胞分离与培养
一旦立项获得批准，接下来就是进行干细胞的分离与培养工作。
这一
过程需要严格控制实验环境，确保干细胞的纯度和活力。
需要优化培
养基配方，探索最适合干细胞增殖和扩增的条件，以确保后续研发工
作的顺利进行。
3. 质量标准的制定与验证
在进行干细胞药物开发的过程中，制定并验证质量标准是至关重要的一环。
这涉及到药物的纯度、活性、稳定性等方面的要求，需要与国家相关的法规和标准相一致。
需要建立完善的质量控制体系，确保生产的每一批药物都符合预定的标准。
4. 临床试验
接下来，就是进行临床试验的环节。
这一阶段需要遵循国家相关的法规和伦理要求，确定试验方案，招募合适的患者，进行安全性和有效性的评估。
临床试验的结果将直接影响后续的药物注册和上市申请，因此需要高度重视。
5. 安全性评估
在药物开发的过程中，安全性评估是至关重要的一环。
这需要进行动物试验，评估药物在体内的毒性和致病性，确保药物的安全性。
需要监测药物可能对人体造成的不良反应，并及时进行调整和改进。
6. 上市申请
当所有前期工作完成之后，就是进行药物上市申请的阶段。
这需要提交完整的临床试验数据和其他相关资料，经过国家相关部门的审核和
批准，方可获得药物上市许可。
需要建立完善的售后监测体系，跟踪药物的使用效果和安全性。
通过以上介绍，相信读者对我国间充质干细胞药物开发流程有了一定的了解。
值得注意的是，干细胞药物的研发过程中存在着诸多的挑战和风险，需要有关部门、企业和科研团队共同努力，确保研发工作的顺利进行，并为人类健康事业作出更大的贡献。
7. 生产工艺
在获得药物上市许可后，接下来是生产工艺的优化和实施。
为了大规模生产符合质量标准的间充质干细胞药物，需要建立起完善的生产工艺。
这包括原料采购、生产设备的选择和优化、生产流程的设计和标准化等工作。
需要建立起完善的质量管理体系，监控整个生产过程的各个环节，确保每一批药物都符合规定的质量标准。
8. 市场推广与应用
随着药物的生产工艺的逐步完善，接下来就是进行市场推广与应用。
这需要制定营销策略，推动药物的推广和应用。
同时需要进行相关学术和临床会议的宣传，以及与医疗机构、保险机构的合作，让更多的患者受益于该项治疗技术。
9. 后续监测
一旦药物上市，后续监测工作也是至关重要的一环。
需要跟踪监测药物在临床应用中的效果和安全性，并进行定期的评估和汇报。
需要借助于大数据分析工具，对临床数据进行深入的分析和挖掘，以进一步完善药物的治疗适应症和用药指南。
10. 技术创新与进步
在药物上市后，相关科研团队也需要不断进行技术创新和进步。
这需要不断深入研究间充质干细胞的性质和机制，探索更有效的治疗方法和途径。
也需要进行相关技术的改进和创新，以提高药物的生产效率和治疗效果，为患者提供更优质的医疗服务。
11. 国际合作和交流
除了在国内进行相关研究和开发工作外，国际合作和交流也是至关重要的。
这不仅有助于获取最新的科研成果和技术进展，还可以推动我国间充质干细胞药物在国际上的认可和推广。
也需要积极参与国际间充质干细胞药物的研发标准和规范的制定，为我国相关产业的发展做出更大的贡献。
12. 伦理和法规的遵守
在整个药物研发、上市和应用的过程中，伦理和法规的遵守都是非常
重要的。
需要严格遵守相关法律法规和伦理规范，确保研发和应用的过程合法合规；另也需要积极参与相关的伦理和法规制定工作，推动相关法规的不断完善和发展，为整个产业的健康发展创造良好的环境和条件。
在总体上，我国间充质干细胞药物的开发流程包括了研发立项、干细胞分离与培养、质量标准的制定与验证、临床试验、安全性评估、上市申请、生产工艺、市场推广与应用、后续监测、技术创新与进步、国际合作和交流以及伦理和法规的遵守等多个环节。
这些环节互相关联、相互促进，构成了一个完整的药物开发和应用生态系统。
在这一生态系统中，各个环节各司其职，共同推动间充质干细胞药物的研发和应用不断向前发展。
同时也要意识到，干细胞药物研发具有一定的挑战和风险，需要有关部门、企业和科研团队共同努力，确保研发工作的顺利进行，并为人类健康事业作出更大的贡献。
希望未来随着科技的持续进步和政策的不断优化，我国的间充质干细胞药物研发和应用能够取得更大的突破和成就。