小鼠无精子症动物模型的构建

目的:建立一种稳定的小鼠无精子症动物模型。方法:60只清洁级C5BL/6小鼠,分为化疗组(1次性腹腔注射白消安10mg/kg体重、环磷酰胺120mg/kg体重)、激素组(每日皮下注射苯甲酸雌二醇40mg/kg,连续注射15d),每组30只。注射后4、12、20周,观察小鼠睾丸生精小管结构的变化以及终止干预后生精功能恢复的情况。结果:化疗组化疗后第4周,睾丸生精小管内精子及精子细胞消失,20周时仍保持无生精状态;激素组连续注射苯甲酸雌二醇后第4周,生精小管内精子及精子细胞消失,但20周时部分生精小管内可以找到精子细胞和精子。化疗组小鼠睾丸重量明显低于激素组(P<0.05)。结论:经过化疗处理的小鼠无精子症模型稳定,雌二醇皮下注射的无精子症模型形成慢而不稳定。相对于雌激素注射方法,化疗是构建小鼠无精子症动物模型比较可靠的方法。     

移植大鼠生精干细胞到裸鼠生精小管实验系统的建立

目的 建立移植大鼠生精干细胞到裸鼠生精小管的实验系统.方法 采用一次腹腔注射busulfan 30mg/kg以消除裸鼠生精小管内源性生精细胞,制备生精干细胞移植受体小鼠;采用laminin粘附的方法分离大鼠生精干细胞;应用生精小管直接注射和输出管注射的方法移植分离到的大鼠生精干细胞进入受体裸鼠的生精小管内.结果 busulfan腹腔注射4周后受体裸鼠生精小管中的精子发生明显得到抑制.大鼠生精干细胞移植2～3个月后在受体裸鼠的生精小管中发现了大鼠的长型精子细胞.结论 成功建立了移植大鼠生精干细胞到受体裸鼠生精小管的试验系统.     

高温热应激构建小鼠精原干细胞移植受体模型

目的:探讨高温热应激方法构建小鼠精原干细胞(SSCs)移植受体的可行性。方法:将4周龄的C57BL/6雄鼠和B6(Cg)-Tyrc-2J/J毛色基因纯合突变雄鼠置于恒温箱内,43℃热应激处理1 h,通过HE染色、免疫组化染色和TUNEL凋亡检测,寻找最佳移植时间;随后将SSCs移植入小鼠生精小管,定期观察移植后受体小鼠睾丸内SSCs增殖、分化及形成精子的情况,并将受体小鼠与正常同周龄雌鼠交配,观察其后代表观遗传学特征。结果:高温热应激3～5 d后受体小鼠睾丸生精小管内生精细胞层数减少,排列紊乱、疏松且大量缺失,间质细胞数量明显减少,生精细胞凋亡明显,自噬水平升高,12 d左右基本恢复。在分离纯化后的SSCs移植8周后,受体小鼠睾丸生精小管内分化形成生精细胞及具有遗传功能的精子,经过自然交配产生正常的后代。结论:高温热应激方法可快速、高效构建小鼠精原干细胞移植受体模型。     

不同剂量中药单体复方对~(60)Co-γ致雄性小鼠生精功能障碍的治疗作用

目的 观察不同剂量中药单体复方(由白藜芦醇、枸杞多糖和槲皮素3种中药单体及L-肉碱组成)促进~(60)Co-γ致小鼠睾丸生精功能障碍的恢复作用,并探讨其最佳剂量.方法 72只雄性昆明小鼠随机分为5组,A组(n=12)正常喂饲,B、C、D、E组(各15只)小鼠均采用~(60)Co-γ 60y全身均匀照射I次,构造小鼠睾丸生精障碍模型,1周后C、D、E组分别用40、80、160mg/kg·d~(-1)浓度的中药单体复方混悬液灌胃35d,A组和B组小鼠用等量生理盐水灌胃.记录小鼠的一般状况、体征及体重变化,于灌胃结束24h后处死,测量小鼠体重、睾丸重量及睾丸指数;酶联免疫法测定血清FSH、LH、T和E_2水平并计算T/E_2比值;并对睾丸组织进行组织学观察.结果 D组(80mg/kg·d~(-1))小鼠体重、睾丸重量及睾丸指数均较B组有明显改善(p<0.05),接近A组(p>0.05),C组和E组虽有所改善但仍低于A组(P<0.05).C、D、E组小鼠血清生殖激素水平均有一定程度的恢复,但D组各生殖激素指标均接近A组(P>0.05).组织学观察见各给药组睾丸生精小管生精细胞层数、精子数目及c.kit阳性表达均高于B组,尤以D组(80mg/kg·d~(-1))恢复最为明显.结论 由白藜芦醇、枸杞多糖、槲皮素和L.肉碱组成的中药单体复方制剂能够促进~(60)Co-γ致小鼠睾丸生精功能障碍后生精功能的恢复,且最佳剂量为80mg/kg·d~(-1) .     

白藜芦醇对2,5-己二酮导致SD大鼠睾丸生精障碍治疗作用的实验研究

目的:观察白藜芦醇促进2,5-己二酮(2,5-HD)所致睾丸生精障碍后的生精功能恢复作用。方法:40只雄性SD大鼠随机分为5组(每组8只),A组正常喂饲,B、C、D、E组均饮用含1%2,5-HD的水溶液5周,然后C、D、E组用不同浓度的白藜芦醇[分别为20、40、80 mg/(kg.d)]治疗9周。比较各组外表体征、体重增加值、睾丸重量的变化,及各组生精小管数目、直径以及生精细胞膜c-kit蛋白表达水平。结果:与A组相比,B、C、D、E组大鼠体质瘦弱、皮肤松弛、毛色暗淡、体重增长减慢、睾丸萎缩;睾丸组织HE染色及免疫组化染色显示生精上皮萎缩,生精细胞发育停滞,c-kit蛋白表达受到抑制。经过白藜芦醇治疗后,C、D、E组大鼠外表体征得到恢复,接近A组,体重和睾丸重量均较B组有所恢复(P<0.01);睾丸生精功能部分恢复,但生精小管数目、直径低于A组水平(P<0.001),同时c-kit蛋白重新获得表达,但低于A组水平(P<0.01)。随着白藜芦醇用量增加,C、D、E组大鼠睾丸生精小管数目、直径显著恢复(P<0.01),生精细胞膜c-kit蛋白表达量增加更为明显(P<0.01)。结论:白藜芦醇可以促进2,5-HD所致大鼠睾丸生精障碍的生精功能恢复。     

小鼠睾丸消化细胞异体移植重构生精小管的研究

目的:采用免疫缺陷小鼠作为受体,通过对小鼠睾丸消化细胞异位移植后不同时期移植物的研究,观察生精小管重构、生精细胞归巢及精子发生情况。方法:取新生ICR小鼠的睾丸消化成单细胞悬液,将其与Matrigel基质胶混匀后移植于雄性裸鼠背部皮下,术后裸鼠行去势。移植后分别于4、6、8、10周处死5只裸鼠,计算移植成功率,取移植物测量直径,并进行HE染色和免疫组化检测,观察生精小管的重构、生精细胞归巢及精子发生情况。结果:20只受体鼠接受睾丸消化细胞移植后全部存活。睾丸消化细胞移植后10周内可见明显隆起的包块,包块直径由第4周的(3.91±0.71)mm增加到(6.69±0.50)mm,移植物表面有血管生成。对移植物石蜡切片进行HE染色可见生精小管样结构,部分生精小管管腔内可见由精原细胞发育至精子细胞的各级生殖细胞,未见明显精子产生。对8周移植物进行免疫组化观察,可见生殖细胞标志物Mvh、支持细胞标志物Gata4和间质细胞标志物P450Scc表达。结论:新生小鼠睾丸消化细胞移植于裸鼠背部皮下后可重构生精小管,为研究睾丸组织工程及睾丸发育和精子发生过程中睾丸各组成细胞之间的相互作用提供了理想的研究模型。     

大鼠骨髓间充质干细胞对雄性生殖系统的修复作用

目的:探讨大鼠骨髓间充质干细胞(BMSCs)在无精子症大鼠模型中对睾丸内环境的修复能力。方法:将同种异体BMSCs移植入无精子症模型大鼠睾丸生精小管中,注射后30 d HE染色观察生精小管细胞组成和结构,免疫组化检测CD44、CD106和c-kit表达情况。结果:与正常大鼠相比,20 mg/kg白消安组中大鼠附睾中精子数量明显减少(P0.01)。分离得到的BMSCs表达CD44和CD106,而不表达c-kit。BMSCs注射移植30 d后,HE染色显示移植组生精小管内形成新的细胞,部分细胞表达CD106,部分细胞表达生殖细胞表面标记c-kit。结论:BMSCs在移植组无精子症大鼠生精小管中能够分化为生殖细胞,对受损的不育大鼠生精小管进行修复。     

射线损伤对睾丸Claudin-11 mRNA表达的影响

目的:Claudin-11为支持细胞紧密连接的组分,在构建血睾屏障及维持生精上皮空间构象中呈现重要作用。本研究拟通过射线局部照射致睾丸氧化应激,观察Claudin-11转录水平变化,探讨氧化应激损伤精子发生的作用机制。方法:48只雄性昆明小鼠随机分配至A、B、C、D 4组,每组12只,A组空白对照,B、C、D 3组分别以2、6、10 Gy剂量60Co-γ射线局部照射实验动物下腹部1次,于4周后处死,测定实验小鼠体重及双睾丸重量;HE染色观察睾丸组织学变化;酶联免疫法检测血清性激素水平;实时荧光PCR监测睾丸组织内抑制素βB及Claudin-11转录水平变化。结果:不同剂量射线致睾丸氧化应激损伤后睾丸重量,A组为(182.9±8.43)mg,B组为(129.4±10.81)mg,C组为(87.5±16.83)mg,D组为(56.1±12.36)mg,呈下降趋势,与A组相比,差异有显著性(P<0.05);睾丸指数A组为(4.28±0.31)mg/g,B组为(3.39±0.57)mg/g,C组为(2.46±0.46)mg/g,D组为(1.63±0.44)mg/g,下降更为明显,与A组相比,差异有显著性(P<0.01);组织学分析示,与A组相比,受射线照射3组生精小管分化指数(TDI)下降明显(P<0.01),生精小管直径减小,生精上皮高度显著降低并层次紊乱。血清FSH,A组为(5.77±1.62)IU/L,B组为(6.74±1.95)IU/L,C组为(8.41±2.44)IU/L,D组为(10.93±3.16)IU/L,逐渐升高,D组较A组升高1.9倍。伴随照射睾丸射线剂量增加,组织内抑制素βB mRNA含量下降,Clau-din11转录水平呈现增高趋势,C、D两组Claudin-11 mRNA水平高于正常对照A组(P均<0.01)。结论:射线局部照射致睾丸氧化应激,组织内抑制素βB mRNA降低、血清FSH升高,支持细胞合成及分泌功能受损;同时,睾丸组织Claudin-11表达量则增加。增加的紧密连接组分同升高的FSH致使血睾屏障重建周期延长,生精上皮内处于减数分裂的精母细胞数量减少,导致不育发生。     

受体性别及完整性对睾丸组织移植物发育的影响

目的以免疫缺陷小鼠为受体,探讨受体的性别及完整性对未成熟睾丸组织移植物生长发育的影响。方法将作为受体的免疫缺陷小鼠分为4组:雄性正常组、雄性去势组、雌性正常组和雌性去势组。移植供体组织均为新生1～2d的昆明小鼠睾丸,移植部位为受体背部皮下。移植7周后取材,计算移植物的回收率并称重,对各组移植物中生精小管结构和生精细胞的组成情况以及生精细胞的染色体进行观察分析。结果移植7周后,从4组受体中获得的移植物体积和重量与移植前相比均有明显增加。染色体观察显示,所有4组移植物中生精细胞染色体数量均为正常2倍体(40条)和单倍体(20条);HE染色结果显示,所有4组受体取出的移植物中均发现带有长形精子的生精小管,其中雄性正常组、雄性去势组、雌性正常组和雌性去势组分别有1、5、1和3只受体中观察到含有长形精子的生精小管。结论新生昆明小鼠睾丸组织分别移植到去势的、未去势的雄性和雌性免疫缺陷小鼠背部7周后,未成熟的生精细胞均可发育成为长形精子。     

小鼠异体Sertoli细胞诱导胰岛移植免疫耐受的作用

目的 观察小鼠Sertoli细胞是否能在异体内起到诱导局部免疫耐受、保护共移植异体胰岛的作用.方法 以糖尿病C57小鼠作移植受体,随机分4组,每组6只;以正常BALB/C小鼠为胰岛供体,正常C57小鼠和正常BALB/C小鼠各作为Serloli细胞供体.A组:单纯移植异体胰岛;B组:移植来源于C57小鼠的Sertoli细胞+BALB/C小鼠来源的胰岛;C组:移植均来源于BALB/C小鼠的Sertoli细胞及胰岛;D组:假手术组.监测各组移植受体的血糖尿糖变化,观察移植物的存活时间.结果 A组移植物平均存活时间为(6.50±2.35)d;B组为(55.67±4.84)d;C组为(51.33±5.05)d;D组未观察到血糖正常.B组及C组的移植方式均可逆转糖尿病小鼠的高血糖状态,移植物存活期均较A组有明显延长,其差异有统计学意义(P<0.05);而B组与C组的移植物存活时间差异无统计学意义(P>0.05).结论 同种异体来源的睾丸Sertoli细胞在异体内可起到诱导局部免疫耐受的效果,对共移植同种异体胰岛起到保护作用,其效果与自体睾丸Sertoli细胞相当.     

Gossypol with methyltestosterone and ethinylestradiol male does not affect rat spermatogonial stem cell differentiation

The purpose of this study was to investigate whether administration of the regimen of gossypol at 12 mg/kg/day combined with methyltestosterone at 20 mg/kg/day and ethinylestradiol at 100 microg/kg/day for a long term of twenty-four weeks could affect the existence and differentiation of rat spermatogonial stem cell. This was assessed by conducting TdT-mediated dUTP nick end-labeling detection, spermatogonial stem cell transplantation and fertility recovery evaluation. Our results showed that spontaneous apoptosis was observed in normal rats' testes from the control group with an apoptotic index (AI) average of 10.24+/-1.52. In the regimen-treated group, the predominant apoptotic cells were spermatocytes and spermatids in the seminiferous tubules. Spermatogonia were not apoptotic (AI averaged 113.42+/-13.24). Two to three months after transplantation of spermatogonial stem cells isolated from regimen-treated rats into recipient nude mice, elongated rat spermatids were identified in the seminiferous tubules of recipient nude mice. Six weeks after withdrawal of the administration, fertility of the regimen-treated rats was recovered compared with that of the control group. The number of litters produced by females mated with regimen-treated males averaged 9.88+/-0.166 matched 10.30+/-0.171 of control group and the litters of the first generation appeared to be normal. These results indicated that the administration of this regimen did not affect the existence and differentiation potential of spermatogonial stem cells of the regimen-treated rats.     

Hematopoietic stem cell transplantation for beta-thalassemia major: experience in south of Iran

INTRODUCTION: Allogeneic stem cell transplantation as a curative treatment for thalassemia major was established in Shiraz in 1993. In this article we describe our results of 10 years experience with allogeneic bone marrow transplantation for thalassemia major. METHODS: From June 1993 to January 2003, 112 cases of beta-thalassemia major underwent allogeneic marrow transplantation from HLA-identical or one antigen-mismatched related donors. Conditioning chemotherapy included busulfan (14 to 15 mg/kg), cyclophosphamide (200 mg/kg), and antithymocyte globulin (ATG; 40 mg/kg). Prophylaxis for graft-versus-host disease consisted of cyclosporine, prednisolone, and methotrexate. RESULTS: One hundred twelve patients with a diagnosis of beta-thalassemia major underwent allogeneic marrow transplantation during this period. The mean age of the patients was 9.5 years with the range of 2 to 20 years. The distribution of cases according to the Lucarelli classification were: 27 cases class I, 38 cases class II, and 47 cases class III. Eighty-seven of 112 patients (77.6%) with diagnosis of beta-thalassemia major are living with full engraftment at a median follow-up of 6 years (range 2 to 119 months). CONCLUSION: Allogeneic bone marrow transplantation has changed the outcome of disease dramatically. According to our results stem cell transplantation is the treatment of choice for class I and II (Lucarelli risk groups). Also, we recommend transplantation as a curative method for treatment of class III beta-thalassemic patients.     

精原细胞异基因移植研究进展及其面临的困惑

将供体精原细胞移植入异基因受体生精小管中,可看到供体精原细胞分化形成供体形态特征的精子。伴随着冷冻保存、体外培养和干细胞富集技术的发展,精原细胞异基因移植必将在基础科学、临床医学、动物繁殖等领域有非常重要的应用前景。另外,这些技术也可用于接受大剂量放疗或化疗的肿瘤患者的性腺保护和生育力保存。本文综述精原细胞异基因移植研究进展,并对该技术在今后临床应用中所存在的问题进行讨论。     

L-肉碱在奥硝唑所致大鼠睾丸和附睾损伤中的保护作用

目的:探讨L-肉碱(LC)在奥硝唑(ORN)所致大鼠附睾和睾丸损伤中的的保护作用。方法:40只雄性SD大鼠(200～230g)随机均分为5组:①A组:给予0.5%的羧甲基纤维素钠(溶剂)灌胃;②B组:每天给予400mg/kgORN灌胃;③C组:每天给予800mg/kgORN灌胃;④D组:每天给予[ORN(400mg/kg)+LC(100mg/kg)]灌胃;⑤E组:每天给予[ORN(800mg/kg)+LC(100mg/kg)]灌胃。上述各组均连续灌胃20d,末次给药24h后,所有大鼠麻醉后处死,分别取睾丸、附睾,进行称重和HE染色,计算睾丸、附睾系数并观察睾丸和附睾病理组织学改变。结果:①与A组相比,B组睾丸、附睾系数明显降低(P<0.05);而C组睾丸、附睾系数为极显著性降低(P<0.01);D组与A组相比无差异,E组与A组相比有极显著性差异(P<0.01);②HE染色显示,与A组相比,B组睾丸生精小管内各级生精细胞排列基本整齐,部分生精小管管腔内有脱落的生精细胞,附睾管腔中精子数目下降,有时可见散在的生精细胞;C组大鼠睾丸生精小管管腔内均可见坏死脱落的生精细胞,附睾管腔中精子数目明显减少,且有较多的非精子细胞成分。D组睾丸生精小管无明显改变,附睾管腔中精子数目也未见明显下降;E组睾丸生精小管管腔内精子数目减少,可见坏死脱落的生精细胞,附睾腔中精子数目明显减少,并伴有较多的非精子细胞成分。结论:奥硝唑(ORN)可导致雄性大鼠附睾和睾丸病理组织学改变,LC对ORN引起大鼠附睾和睾丸损伤具有一定的保护作用。     

Transplantation of human peripheral blood stem cells into fetal rhesus monkeys (Macaca mulatta)

BACKGROUND: Methods for assessing engraftment efficiency have been explored in a primate xenogeneic model of in utero hematopoietic stem cell transplantation. METHODS: Human peripheral blood stem cells (PBSC) were obtained by leukapheresis from a human male donor after 4 days of administration of recombinant human granulocyte-colony stimulating factor (5 microg/kg/ day). PBSC were enriched for the CD34+ population with and without T-cell depletion. The resulting mononuclear cells consisted of two cell populations, one that was stem cell enriched (0.83% CD3+ cells, 95% CD34+; group 1) and one that was stem cell enriched and T-cell depleted (<0.03% CD3+ cells, 98% CD34+; group 2). Four fetal monkeys (two per group) received either two or four i.p. injections (approximately 5x10(6) cells/injection) via ultrasound guidance every other day over a 7-day period (gestational days 50, 52, 54, and 56). One fetus in each group also received i.p. recombinant human stem cell factor (25 microg/kg) and recombinant human granulocyte-colony stimulating factor (10 microg/kg) posttransplant every 10 days from gestational day 60-150. RESULTS: Four healthy newborns were delivered at term, and specimens were analyzed by polymerase chain reaction for the human Y chromosome (birth, monthly to 6 months; blood, marrow, progenitor assays). Polymerase chain reaction results were positive for all four newborns in all specimens assessed, and flow cytometric analysis for human CD45 in marrow showed engraftment ranging from 0.1-1.7%. There was no evidence of graft-versus-host disease in any of the animals. CONCLUSION: These studies show that (1) multilineage engraftment of human PBSC can be achieved in the fetal rhesus recipient, (2) the rhesus fetus appears to tolerate relatively high numbers of human CD3+ cells, and (3) healthy chimeric rhesus infants can be delivered at term after multiple in utero procedures.     

Micafungin Does Not Influence the Concentration of Tacrolimus in Patients After Allogeneic Hematopoietic Stem Cell Transplantation

Background

Tacrolimus is commonly used in stem cell transplant recipients for prophylaxis of graft-vs-host disease. Micafungin is widely used as a strong antifungal agent in empirical therapy in patients with febrile neutropenia. Both tacrolimus and micafungin are substrates of cytochrome P450 3A4 in vitro. Therefore, there is risk of drug interaction with concomitant administration of these drugs.

Objective

To estimate the drug interaction of tacrolimus and micafungin by evaluating the pharmacokinetics in 6 patients who had undergone allogeneic stem cell transplantation.

Results

The mean (SD) concentration-dose ratio of tacrolimus in all patients at 1, 4, 8, and 24 hours after concomitant administration of micafungin was 607 ± 306, 653 ± 328, 699 ± 340 and 671 ± 403 (ng/mL)/(mg/kg/d), respectively, and without micafungin was 756 ± 314 (ng/mL)/(mg/kg/d). The percentage of the concentration-dose ratio in patients treated with tacrolimus and micafungin vs patients treated with tacrolimus alone was 98%, 105%, 112%, and 108% at 1, 4, 8, and 24 hours, respectively. For both tacrolimus and micafungin, the 90% confidence intervals for the primary pharmacokinetic parameters (ie, the concentration-dose ratio at each point) ranged from 80% to 125%.

Conclusion

We conclude that there is no drug interaction between tacrolimus and concomitantly administered micafungin in stem cell transplantation recipients.     

Vascular Endothelium Changes After Conditioning in Hematopoietic Stem Cell Transplantation: Role of Cyclophosphamide and Busulfan

Vascular endothelial injury, a feature of some complications of hematopoietic stem cell transplantation (HSCT), is characterized by increased endothelial cells. We investigated that classical pretreatment drugs in HSCT could result in vascular endothelial injury in mice. Six-to eight-week-old female BALB/c mice were divided into a control group, a cyclophosphamide group (60 mg/kg per day for 2 days) and a busulfan group (4 mg/kg per day for 4 days). We observed the general state of health and regularly counted the number of white blood cells. Circulating endothelial cells and their progenitors were estimated by flow cytometry. Morphologic endothelial changes were analyzed with optical and transmission electron microscopy. After conditioning with cyclophosphamide or busulfan, white blood cells fell to a low number with injuries noted on hematoxylin and eosin-stained pathology sections. Circulating endothelial cells and their progenitors peaked significantly higher than in the control group. Vascular endothelial injuries were observed in the 2 experimental groups by transmission electron microscopy. These data support the hypothesis the vascular endothelial injury occurs during conditioning with cyclophosphamide or busulfan for HSCT, with simultaneous mobilization of endothelial progenitor cells.     

Transplantation of reproductive organs

A historical review of the literature concerning replantation and transplantation of reproductive organs has included studies from this laboratory, using rats, over the past 25 years. From the basic observation of ischemic and traumatic injury due to the transplantation, syngeneic testicle transplants, resulting in a partner's impregnation and histological restoration of the testicles, led to human testicular transplantation. As to the ovarian transplants, granulosa-theca cell tumors may transform into malignancies if followed for a prolonged period as intrasplenic ovaries, and high doses (15 to 20 mg/kg b.w.) of azathioprine can produce such malignant tumors in a shorter period. By caval-portal shunt, ovarian hormones enter directly into the portal blood stream and no typical granulosa-theca cell tumors were produced, owing to the fact that the liver cannot degrade all the hormones secreted by both ovaries. While en-bloc vagino-utero-ovarian transplantation in the rat is possible, no impregnation has been yet achieved. Finally, it is hypothesized that those who have acquired microsurgical techniques and have a full understanding of the anatomy of the reproductive system will not only be able to perform replantation of the penis, but also will be capable of allogeneic transplantation of genital organs, whether ethically approved or not, and sooner than one may think. In such cases a penile part may be obtained at a sex-change surgery or from a cadaveric donor, similar to other vital organ transplantation practices. © 1995 Wiley-Liss, Inc.     

New Culture Medium Concepts for Cell Transplantation

Background

Before cell or tissue transplantation, cells or tissues have to be maintained for a certain period in vitro using culture medium and methods. Most culture media contain substances such as pH indicators and buffers. It is not known whether some of these substances are safe for subsequent application in the transplantation of cells or tissues into the human body. We investigated culture media and methods with respect to the safety of the components in future transplantation applications.

Methods

A modified culture medium—medical fluid-based culture medium (FCM)—was designed by using various fluids and injectable drugs that are already currently permitted for use in clinical medicine. Medium components necessary for optimal cell growth were obtained from approved drugs. FCM was manufactured with adjusted final concentrations of the medium components similar to those in commercial Dulbecco's modified Eagle's medium (DMEM). In particular, 1029.40 mg/L amino acids, approximately 88.85 mg/L vitamins, 13,525.77 mg/L inorganic salts, and 4500 mg/L D-glucose comprise the high-glucose FCM. Next, human fat synovium-derived mesenchymal stem cells and rat H9c2 (2-1) cells were cultured under 2 conditions: (1) DMEM-high glucose (HG), an original commercial medium, and (2) optimized FCM-HG. We assessed the morphologies and proliferation rates of these cells.

Results

We observed that FCM-HG was able to induce the growth of FS-MSC and commercially available H9c2 cell. The morphologies and proliferation patterns of these cells cultured under FCM-HG showed no differences compared with cells grown in DMEM-HG.

Conclusion

Our data suggest that FCM, which we developed for the first time according to the concept of drug repositioning, was a useful culture medium, especially in cultured cells intended for human cell transplantation.