果糖和二硫苏糖醇对骨髓间充质干细胞冻存后活性及多能性的影响

背景:冻存是保证干细胞临床应用的关键步骤之一,但现有冻存技术常导致细胞活性降低、多能性丧失及分化能力下降。目的:探究果糖及二硫苏糖醇是否有助于维持冻存后骨髓间充质干细胞多能性及成骨分化潜能。方法:分离培养SD大鼠骨髓间充质干细胞,在细胞冻存前分别用果糖(200μmol/L),二硫苏糖醇(500μmol/L)μmol/L)及果糖(200μmol/L)+二硫苏糖醇(500预处理1 h。冻存6个月后,复苏细胞并用倒置显微镜观察细胞形态,MTT实验检测细胞活性,定量PCR检测相关干性基因(Nanog,Oct4及Sox2)的表达,碱性磷酸酶活性测试及茜素红染色检测复苏骨髓间充质干细胞成骨分化能力。结果与结论:(1)复苏后各组细胞在形态上无明显差别;(2)果糖预处理及联合预处理有助于骨髓间充质干细胞活性维持;(3)二硫苏糖醇预处理可显著促进骨髓间充质干细胞多能性相关基因Nanog及Sox2的表达;(4)果糖、二硫苏糖醇及联合预处理皆有助于维持骨髓间充质干细胞成骨分化潜能,但以二硫苏糖醇及联合预处理组效果最佳;(5)结果表明,果糖预处理有助于维持冻存骨髓间充质干细胞活性,二硫苏糖醇有助于维持冻存骨髓间充质干细胞多能性及成骨分化能力。     

人前脑啡肽基因修饰骨髓间充质干细胞系的生物学特性

BACKGROUND:Previous studies have found that the transplantation of modified cell lines exhibit analgesic effect. But little is reported on the biological characteristics of human preproenkephalin gene-modified bone marrow mesenchymal stem cell lines. OBJECTIVE:To observe the biological characteristics of human preproenkephalin gene-modified bone marrow mesenchymal stem cells. METHODS:Bone marrow mesenchymal stem cells were isolated and cultured to establish human preproenkephalin gene-modified bone marrow mesenchymal stem cell lines. RESULTS AND CONCLUSION:After freezing-thawing, passage 4 human bone marrow mesenchymal stem cells, human bone marrow mesenchymal stem cells-pBABE and human bone marrow mesenchymal stem cells-human preproenkephalin exhibited no significant changes in the cell viability (P > 0.05), as well as in the fat cell proportion after adipogenic induction (P > 0.05). Moreover, red calcium deposition was presented in all these cells by alizarin red staining after osteogenic induction. Flow cytometry results showed that passage 4 human bone marrow mesenchymal stem cells, human bone marrow mesenchymal stem cells-pBABE and human bone marrow mesenchymal stem cells-human preproenkephalin could express CD29 and CD44, but not express CD34 and CD45. Human preproenkephalin genes were highly expressed in human bone marrow mesenchymal stem cells-pBABE, but lowly expressed in passage 4 human bone marrow mesenchymal stem cells. Additionally, recombinant plasmid pBABE-preproenkephalin-modified human bone marrow mesenchymal stem cells could express enkephalin protein. In conclusion, human preproenkephalin gene-modified human bone marrow mesenchymal stem cell lines can maintain the pluripotent differentiation or proliferation capacity of bone marrow mesenchymal stem cells, and secrete enkephalin protein.     

Evaluation of bone marrow-derived mesenchymal stem cells after cryopreservation and hypothermic storage in clinically safe medium

Achievements in tissue engineering using mesenchymal stem cells (MSC) demand a clinically acceptable "off-the-shelf" cell therapy product. Efficacy of cryopreservation of human bone marrow-derived MSC in clinically safe, animal product-free medium containing 2%, 5%, and 10% dimethyl sulfoxide (DMSO) was evaluated by measuring cell recovery, viability, apoptosis, proliferation rate, expression of a broad panel of MSC markers, and osteogenic differentiation. Rate-controlled freezing in CryoStor media was performed in a programmable cell freezer. About 95% of frozen cells were recovered as live cells after freezing in CryoStor solutions with 5% and 10% DMSO followed by storage in liquid nitrogen for 1 month. Cell recovery after 5 months storage was 72% and 80% for 5% and 10% DMSO, respectively. Measurements of caspase 3 activity demonstrated that 15.5% and 12.8% of cells after 1 month and 18.3% and 12.9% of cells after 5 months storage in 5% and 10% DMSO, respectively, were apoptotic. Proliferation of MSC recovered after cryopreservation was measured during 2 weeks post-plating. Proliferation rate was not compromised and was even enhanced. Cryopreservation did not alter expression of MSC markers. Quantitative analysis of alkaline phosphatase (ALP) activity, ALP surface expression and Ca?? deposition in previously cryopreserved MSC and then differentiated for 3 weeks in osteogenic medium demonstrated the same degree of osteogenic differentiation as in unfrozen parallel cultures. Cell viability and functional parameters were analyzed in MSC after short-term storage at 4°C in HypoThermosol-FRS solution, also free of animal products. Hypothermic storage for 2 and 4 days resulted in about 100% and 85% cell recovery, respectively, less than 10% of apoptotic cells, and normal proliferation, marker expression, and osteogenic potential. Overall, our results demonstrate that human MSC could be successfully cryopreserved for banking and clinical applications and delivered to the bedside in clinically safe protective reagents.     

冻存脐带间充质干细胞向成骨细胞的分化

背景：在骨组织工程中,脐带间充质干细胞是的一种新兴的种子细胞。目前认为低温冻存是长期保存细胞的有效方法。 目的：探究冻存的脐带间充质干细胞能否被诱导分化成成骨细胞。 方法：采用组织块贴壁法从脐带的华尔通氏胶组织中分离出间充质干细胞。然后,用倒置显微镜观察原代细胞的细胞形态。脐带间充质干细胞的免疫表型和细胞周期用流式细胞仪检测。在冻存6个月后,复苏第2代脐带间充质干细胞进行冻存复苏,并传代培养至12代。对第12代的脐带间充质干细胞进行成骨诱导,它的成骨能力分别通过碱性磷酸酶活性检测,骨钙素和骨涎蛋白的免疫荧光检测以及茜素红染色检测来 确定。 结果与结论：原代脐带间充质干细胞呈现典型的成纤维细胞样形态。流式细胞仪显示培养的细胞高表达间充质干细胞的表面标志CD73、CD105和CD90,但是不表达造血细胞的表面标志CD34和CD45。复苏后细胞的存活率是90%。细胞周期显示P8的细胞有75%处于G₀/G₁期,25%处于S+G₂M期。经成骨诱导液处理的第12代细胞显示出比对照组更高的碱性磷酸酶活性(P < 0.01)。此外,在成骨诱导液中诱导的细胞对骨钙素和骨涎蛋白的染色呈阳性,并形成矿化了结节。冻存后的脐带间充质干细胞仍保持了它们的生物学特性,并且在成骨诱导液中能被诱导分化成成骨细胞。     

Cryopreservation of osteoblast-like cells: viability and differentiation with replacement of fetal bovine serum in vitro

In reconstructive medicine, the clinical use of cryopreservation techniques depends on the absence of infectious agents such as prions. Therefore, we investigated the viability and differentiation of human osteoblast-like cells during replacement of fetal bovine serum in vitro. The aim of the present study is to replace the potentially infectious supplement fetal bovine serum during the cryopreservation procedure in order to perform future clinical trials. We used a cryopreservation technique with Me(2)SO for human osteoblast-like cells of iliac cancellous bone. In the cell culture of cryopreserved and fresh osteoblast-like cells, we substituted Dulbecco's modification of Eagle's medium (DMEM)/Ham's F12 plus 1% penicillin/streptomycin with autologous serum, human serum albumin and Biseko for fetal bovine serum. For the fourth treatment group, we removed fetal bovine serum without replacing it. DMEM/Ham's F12 plus 1% penicillin/streptomycin with fetal bovine serum served as the control group. After 4, 7, 14 and 21 days of culture for the cryopreserved and noncryopreserved cells, we performed cell counting, a WST-1 test, ELISA for collagen type I, and osteocalcin. The activity of alkaline phosphatase was also measured. The best results were obtained for the group with autologous serum as a supplement after thawing, exceeding the other groups with regard to proliferation rate. Most viable cells were observed with no replacement before freezing and after thawing of the cells. With regard to differentiation, the cultures with autologous serum after thawing of the cells showed little concentration of the differentiation markers, probably due to early contact inhibition of the cells in vitro. With regard to effort and outcome, the most promising group for cryopreservation was the one with DMEM/Ham's F12 plus 1% penicillin/streptomycin alone before freezing, especially when osteoblast-like cells were cultured in medium with autologous serum after thawing. This is important, as this in vitro setting resembles the in vivo situation when cryopreserved bone is transplanted. These findings indicate that, for clinical purposes, fetal bovine serum can be removed for cryopreservation of iliac cancellous bone with minor loss of viability.     

多发性骨髓瘤患者骨髓间充质干细胞冻存前后的生物学特性

背景：多发性骨髓瘤患者的骨髓间充质干细胞具有多向分化、免疫调节和支持造血作用,但是这些功能是否受冻存的影响目前尚不清楚。 目的：探讨冻存对多发性骨髓瘤患者骨髓间充质干细胞生物学特性的影响。 方法：采用细胞贴壁法获取多发性骨髓瘤患者骨髓间充质干细胞,将传代后的细胞用IMDM细胞冻存液(含10%的二甲基亚砜和体积分数40%的胎牛血清)保存在-196 ℃液氮中。检测短期(1个月)和长期(12个月)冻存复苏后间充质干细胞的活性和增殖能力;将冻存后多发性骨髓瘤患者骨髓间充质干细胞作为滋养层,应用甲基纤维素半固体培养,检测其支持造血的能力;混合淋巴细胞反应检测冻存后多发性骨髓瘤患者骨髓间充质干细胞调控免疫能力。 结果与结论：经过短、长期冻存后多发性骨髓瘤患者骨髓间充质干细胞的细胞活性分别为(92.9±7.5)%和(86.7±9.2)%;短、长期冻存后细胞的增殖能力与冻存前间充质干细胞相似;冻存后多发性骨髓瘤患者骨髓间充质干细胞仍具有支持造血祖细胞生长的作用和抑制T淋巴细胞增殖的能力,与冻存前相比,没有明显差别。说明冻存可以降低多发性骨髓瘤患者骨髓间充质干细胞的细胞活性,但是并不影响间充质干细胞的增殖、支持造血和免疫调节的能力。     

Long-term cryopreservation of human mesenchymal stem cells using carboxylated poly-l-lysine without the addition of proteins or dimethyl sulfoxide

Human bone marrow-derived mesenchymal stem cells (hBMSCs) are known for their potential to undergo mesodermal differentiation into many cell types, including osteocytes, adipocytes, and chondrocytes. Therefore, hBMSCs can be used for a variety of regenerative medicine therapies, in fact, hBMC-derived osteocytes have already been used in bone reconstruction. This study discusses the viability and the differentiation properties of hBMSCs that have been cryopreserved in the absence of proteins or dimethyl sulfoxide (DMSO) by using a novel polyampholyte cryoprotective agent (CPA). This CPA is based on carboxylated poly-l-lysine (COOH-PLL) and it was produced by a reaction between ?-poly-l-lysine and succinic anhydride. ¹H-NMR and two-dimensional correlation (¹H-¹³C HSQC) spectroscopy revealed that COOH-PLL did not have a special structure in solution. The hBMSCs can be cryopreserved for 24?months at ?80?°C by using a 7.5% (w/w) cryopreserving solution of COOH-PLL, which introduces carboxyl groups that result in?>?90% cell viability after thawing. Furthermore, the cryopreserved hBMSCs fully retained both their proliferative capacity as well as their potential for osteogenic, adipogenic, and chondrogenic differentiation. Confocal laser-scanning microscopy showed that the polyampholyte CPA did not penetrate the cell membrane; rather, it attached to the membrane during cryopreservation. These results indicate that the cryoprotective mechanisms of COOH-PLL might differ from those of currently used small molecule CPAs. These results also suggest that using COOH-PLL as a CPA for hBMSC preservation can eliminate the use of proteins and DMSO, which would be safer if these cells were used for cell transplantation or regenerative medicine.     

间充质干细胞的成骨细胞分化和免疫组织化学鉴定

目的 探讨脐带的间充质干细胞分化为成骨细胞的影响因素和免疫组织化学鉴定。 方法 收集306例冻存的健康胎儿脐带,采用华尔通胶组织块贴壁法从脐带组织中分离间充质干细胞,利用荧光显微镜观察原代细胞的细胞形态。脐带间充质干细胞的免疫表型和细胞周期采用免疫组织化学检测。复苏冻存的脐带间充质干细胞,并传代培养至10代。对第10代的脐带间充质干细胞进行成骨诱导,其成骨能力分别通过钙结节和骨涎蛋白的免疫荧光检测以及茜素红染色检测来确定。 结果 免疫组织化学结果显示,培养的细胞高表达间充质干细胞的表面标志物CD73、CD90和CD105,但是不表达造血细胞的表面标志物CD34和CD45。复苏后细胞的存活率是90%。细胞周期显示,第10代的细胞有80%处于G₀/G₁期,20%处于S+G₂/M期。成骨细胞刺激因子诱导干细胞的骨涎蛋白染色呈阳性,并形成矿化的钙结节。 结论 冻存的脐带间充质干细胞在成骨细胞刺激因子下能被诱导分化为成骨细胞,具有高度自我增殖和多向分化能力。     

低温保存脂肪间充质干细胞的生物学特性评价

目的 探讨低温保存对小鼠脂肪间充质干细胞（ADMSCs)体外培养的生物学特性和分化潜能的影响。方法 分离、培养小鼠ADMSCs,取第3代细胞置于液氮深低温（-196℃）冻存 12个月后复苏。MTT 法测定ADMSCs的增殖活性;β-半乳糖苷酶(SA-β-Gal)染色检测衰老;免疫细胞化学方法检测细胞表面分子CD73、CD90和CD105;条件培养基诱导后,免疫荧光法检测心肌特异性肌钙蛋白（cTnT）,茜素红、碱性磷酸酶和油红 O 染色检测成骨、成脂诱导分化潜能,Real time-PCR检测cTnT、Gata4、Ost和Runx2。未经低温保存的第3代ADMSCs为对照。 结果 低温保存的ADMSCs其增殖活性、衰老率与未冻存细胞比较差异无显著性（P>0.05）。诱导后ADMSCs的cTnT阳性表达、茜素红、碱性磷酸酶和油红O染色呈阳性反应,冻存组与对照组比较差异无显著性(P>0.05）。结论 低温保存对ADMSCs体外生长特性和成心肌、成脂肪细胞、成骨细胞的分化潜能差异无显著性。     

The ROCK Inhibitor Y-27632 Negatively Affects the Expansion/Survival of Both Fresh and Cryopreserved Cord Blood-Derived CD34+ Hematopoietic Progenitor Cells

Cord blood (CB) is an unlimited source of hematopoietic stem and progenitor cells (HSPC). The use of cryopreserved CB-derived CD34+ HSPCs is successful in children and usually leads to rapid hematopoietic recovery upon transplantation. However, current methods for ex vivo expansion of HSPCs still result in a loss of multilineage differentiation potential and current freeze-thawing protocols result in significant cell death and loss of CD34+ HSPCs. The major cause for the loss of viability after slow freezing is apoptosis induced directly by cryoinjury. Very recent reports have demonstrated that Y-27632, a selective and robust ROCK inhibitor is a potent inhibitor of the apoptosis and is efficient in enhancing the post-thaw survival and recovery of different human stem cells including human embryos, hESCs, induced pluripotent stem cells and mesenchymal stem cells. Here, we analyzed the effect of such an inhibitor in CB-derived CD34+ HSPCs. CB-derived CD34+ HSPCs were MACS-isolated and treated with or without 10 μM of Y-27632. The effect of Y-27632 on culture homeostasis was determined in both fresh and cryopreserved CB-derived CD34+ HSPCs. Our results indicate that the Y-27632 not only dramatically inhibits cell expansion of both fresh and cryopreserved CD34+ HSPCs but also impairs survival/recovery of CD34+ HSPCs upon thawing regardless whether Y-27632 is added to both the cryopreservation and the expansion media and or just to the expansion culture medium with or without hematopoietic cytokines. This study identifies for the first time a detrimental effect of Y-27632 on the expansion and survival of both fresh and cryopreserved CB-derived CD34+ HSPCs, suggesting that Y-27632 may have a differential impact on distinct lineage/tissue-specific stem cells. Our data suggest different functions of Y-27632 on human stem cells growing in suspension versus those growing attached to either treated tissue culture plastic or extracellular matrix. We discourage any clinical application of Y-27632 in potential technical developments aimed at improving cryopreservation procedures of CB-derived cells and/or in vitro expansion of HSPCs without spontaneous differentiation.     

体外扩增过程中人骨髓间充质干细胞的增殖与分化规律

目的：系统考察体外扩增过程中人骨髓间充质干细胞（MSC）的增殖与分化规律，为MSC任组织修复以及细胞治疗中的应用提供参考、方法：以全骨髓贴壁法分离成人肋骨骨髓MSC，在相同条件下分别考察各代细胞形态、生长、表面标记、细胞周期、成骨、成软骨及成脂肪能力的变化情况。结果：随代次增加，MSC增殖能力、成骨、成脂肪能力均有所下降，而成软骨能力无明显降低；成骨、成软骨及成脂肪能乃均保持到细胞衰老。存扩增过程中，MSC始终保持较高的纯度，CD29、CD44、CD105的阳性率均在90％以上，CD14、CD34和CD45的阳性率均在4％以下、结论：在体外培养过程中MSC干细胞特性逐渐丢失，其中向骨、脂肪方向的分化潜能较软骨方向更易失去；而多向分化能力的保持较之自我更新能力更为持久。MSC在7代以前可作为基础研究及临床应用的良好对象。     

大鼠骨髓间充质干细胞的纯化、冻存及成瘤性分析

背景：骨髓间充质干细胞在骨髓中的含量极低,体外的长期培养过程中易丧失干细胞潜能以及体内移植后安全性等问题的存在,限制了骨髓间充质干细胞在临床上的广泛应用。 目的：探索体外分离纯化、冻存大鼠骨髓间充质干细胞的最适方法,并观察以此方法培养的骨髓间充质干细胞移植体内后是否具有成瘤性。 方法：分别采用差速贴壁结合24 h首次换液、24 h首次换液、48 h首次换液的方法纯化培养骨髓间充质干细胞,筛选最适纯化方法进行后续实验。配制含体积分数为10%,20%,30%,40%,50%胎牛血清的细胞冻存液冻存细胞,复苏后计算细胞存活率,测定复苏后细胞的生长曲线及成脂诱导能力。将第3,15代骨髓间充质干细胞进行裸鼠肌肉、肝脏局部注射体内移植,45 d后取注射部位行病理组织标本检查。 结果与结论：差速贴壁结合24 h首次换液法所获得骨髓间充质干细胞纯度最高,而细胞增殖能力与其他两组无明显差别,因此选用该方法纯化骨髓间充质干细胞,然后进行传代培养;含体积分数为30%血清冻存液既可保证细胞活性与增殖能力,又可保证干细胞特性及多向分化潜能。骨髓间充质干细胞传至15代仍保持间充质干细胞特性,骨髓间充质干细胞移植入裸鼠肝脏45 d后仍可在肝脏局部存活,生长状态与体外培养相似,无异型性及向周围浸润生长,提示体外长时间培养15代以内的骨髓间充质干细胞可在裸鼠体内存活且无成瘤性。     

Survivability of rabbit amniotic fluid-derived mesenchymal stem cells post slow-freezing or vitrification

This work aimed to evaluate the effect of two distinct cryopreservation procedures - conventional slow-freezing and vitrification, on survivability and mesenchymal marker expression stability of rabbit amniotic fluid-derived mesenchymal stem cells (rAF-MSCs). Cells at passage 2 were slowly frozen, using 10% of dimethylsulfoxide, or vitrified, using 40% of ethylene glycol, 0.5 M sucrose and 18% Ficoll 70. After three months storage in liquid nitrogen, viability, chromosomal stability, ultrastructure, surface and intracellular marker expression and differentiation potential of cells were evaluated immediately post-thawing/warming and after additional culture for 48–72 h. Our results showed decreased (P ≤ 0.05) viability of cells post-thawing/warming. However, after additional culture, the viability was similar to those in fresh counterparts in both cryopreserved groups. Increase (P ≤ 0.05) in the population doubling time of vitrified cells was observed, while doubling time of slow-frozen cells remained similar to non-cryopreserved cells. No changes in karyotype (chromosomal numbers) were observed in frozen/vitrified AF-MSCs, and histological staining confirmed similar differentiation potential of fresh and frozen/vitrified cells. Analysis of mesenchymal marker expression by qPCR showed that both cryopreservation approaches significantly affected expression of CD73 and CD90 surface markers. These changes were not detected using flow cytometry. In summary, the conventional slow-freezing and vitrification are reliable and effective approaches for the cryopreservation of rabbit AF-MSCs. Nevertheless, our study confirmed affected expression of some mesenchymal markers following cryopreservation.     

Factors influencing post-cryopreserved CD34+ cells viability in the harvested products of autologous haematopoietic stem cells

The cryopreservation process of stem cells potentially cause the loss of CD34+ cells. The aim of this study is to evaluate association of patient, graft and technical characteristics with post cryopreserved CD34+ cells viability among lymphoproliferative disease namely multiple myeloma (MM) and lymphoma patients at Hospital Universiti Sains Malaysia (USM). This retrospective study was conducted in the Transplant Unit. A search of the hospital data (2008–2018) to identify 132 patients for both MM and lymphoma who underwent autologous peripheral blood haematopoietic stem cells (APBSC) mobilisation, and were successfully harvested and cryopreserved. Selected patients’ profile as well as selected parameters of stem cell mobilization and cryopreservation were obtained from laboratory information system (LIS), record unit and the Transplant Unit. Multiple logistic regression (MLR) was used to find significant associated factors and P < 0.05 was considered significant. The mean age of the patients was 39 years old with almost equal gender distribution and majority were lymphoma patients, 96 (72.7%) while 36 (27.3%) were multiple myeloma (MM) patients. The significant influencing factors of post-cryopreserved CD34+ cells viability were pre-cryopreserved CD34+ cell viability, total nucleated cells (TNC), and anti-platelet and antibiotics usage. Patients who are not on anti-platelet and have higher pre-cryopreserved CD34+ cells viability have higher chance for good post-cryopreserved CD34+ cells viability. While, those patients with higher TNC and on antibiotics have lower chance for good post cryopreserved CD34+ cells viability. This study showed patients who are not on anti-platelet and antibiotics will have higher probability of achieving good post cryopreserved CD34+ cells viability. The APBSC products with higher pre-cryopreserved CD34+ cells viability and lower TNC will achieve better post-cryopreserved CD34+ cells viability. The addition of extra plasma to the APBSC products is recommended to reduce the TNC.     

豚鼠骨髓间充质干细胞的分离培养及成骨诱导

背景：利用骨髓间充质干细胞的取材灵活性及快捷性,对已掌握的培养技术及成骨诱导进一步探索性研究。 目的：通过建立豚鼠骨髓间充质干细胞的体外分离培养法,探讨豚鼠骨髓间充质干细胞表型特征以及多项分化潜能。 方法：利用贴壁培养法分离纯化豚鼠骨髓间充质干细胞,传代扩增,流式细胞分析检测细胞表面分子CD29、CD44、CD45的表达。分别采用成骨诱导培养液和成脂诱导培养液定向诱导骨髓间充质干细胞向脂肪细胞、成骨细胞分化。 结果与结论：原代分离的骨髓间充质干细胞在接种后96 h贴壁,细胞形态为椭圆形,多角形及短梭形,8 d时细胞呈长梭形并达到90%单层融合。经传代扩增,细胞进一步纯化,细胞形态为均一的长梭形并呈漩涡状排列,而且生长速率加快。流式细胞检测 CD29、CD44阳性率分别为95.7%和65.7%。不同诱导剂定向诱导后,经油红O、茜素红S、碱性磷酸酶染色、免疫组织化学Ι型胶原酶鉴定,P3代骨髓间充质干细胞分别向脂肪细胞及成骨细胞分化。结果表明,通过贴壁筛选方法,体外分离培养的豚鼠骨髓间充质干细胞具有很强的增殖能力,并保持稳定的表型特征及多向分化潜能。     

The effects of freeze/thawing process on cryopreserved equine umbilical cord blood-derived mesenchymal stem cells

The use of mesenchymal stem cells (MSCs)-derived equine umbilical cord blood in cell grafts transplantation is advantageous; therefore, preservation of these cells is of utmost importance in repair therapies. To evaluate the viability ratio of the MSCs obtained from equine umbilical cord blood after cryopreservation, umbilical cord blood obtained from nine 46–48-week-old foals were purified for harvesting MCs. The purified cells were frozen from the first to tenth passages and stored in liquid nitrogen. After thawing, the cell viability was assessed through trypan blue staining procedure. The highest viability (>80%) ratio was observed with the cells derived from the first passage in 1 and 8?weeks after cryopreservation. However, the viability of cells was dependent on the passage used for cryopreservation. Results in this study demonstrated that equine umbilical cord blood stem cells could successfully be frozen and stored in liquid nitrogen for 8?weeks without significant change in the characterization of the cells cryopreserved as regards their viability, growth ability, and differentiation potential.     

CD146阳性亚群有作为软骨组织工程种子细胞的应用潜力

文题释义：CD146：也称为MUC18,MCAM,Mel-CAM或S-Endo1,是一种跨膜糖蛋白,同时属于免疫球蛋白超级家族的一员。在人体组织中,CD146阳性细胞被认为是微血管的壁细胞,其具有较强的增殖、迁移及自我更新能力。 组织工程种子细胞：组织工程包括3个关键要素——种子细胞、支架和活性因子,其中种子细胞应具有良好的细胞活性、增殖能力、分化及合成基质等生物功能。 背景：再生医学的发展、组织工程技术的出现为软骨缺损重建提供了新的解决思路。在组织工程中,间充质干细胞是应用广泛的种子细胞,然而干细胞作为一个异质性的细胞群体其不同亚群发挥着不同的功能。因此,应用间充质干细胞关键功能亚群进行软骨修复具有广泛应用前景。 目的：从人脂肪间充质干细胞中分离出CD146阳性亚群细胞,验证其生物学特性及其作为软骨组织工程种子细胞的潜力。 方法：人脂肪间充质干细胞由浙江金时代生物技术有限公司提供,通过流式细胞术对人脂肪间充质干细胞表面标志物进行鉴定,应用免疫磁珠分选方法从人脂肪间充质干细胞中分选出CD146阳性表达的细胞亚群。通过基因芯片检测技术及生物信息学分析技术揭示2种细胞的分子特性;体外诱导2种细胞成软骨分化并进行鉴定;冻存复苏前后检测2种细胞的细胞活性及凋亡情况。 结果与结论：①人脂肪间充质干细胞表达高水平干细胞相关标志物CD73、CD90,不表达造血干细胞相关标志物CD34、CD45、HLA-DR;②生物信息分析结果表明CD146阳性亚群细胞与脂肪间充质干细胞相比在炎症通路及骨骼肌肉系统疾病有不同功能;③CD146阳性亚群细胞能够成球软骨分化,并且其成软骨分化能力要优于人脂肪间充质干细胞;④CD146阳性亚群细胞复苏后凋亡情况和活性均要优于人脂肪间充质干细胞;⑤结果表明,CD146阳性亚群细胞具有良好的成软骨分化潜力,是一种具有前景的软骨组织工程种子细胞。 ORCID: 0000-0003-4210-4708(眭翔) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Maintenance of phenotype and function of cryopreserved bone-derived cells

The emerging fields of tissue engineering and regenerative medicine require large numbers of cells for therapy. Although the properties of cells obtained from a variety of fresh tissues have been delineated, the knowledge regarding cryopreserved grafts-derived cells remains elusive. Previous studies have shown that living cells could be isolated from cryopreserved bone grafts. However, whether cryopreserved bone-derived cells can be applied in regenerative medicine is largely unknown. The present study was to evaluate the potential application of cryopreserved grafts-derived cells for tissue regeneration. We showed that cells derived from cryopreserved bone grafts could maintain good proliferation activity and osteogenic phenotype. The biological phenotype of these cells could be well preserved. The transplantation of cryopreserved bone-derived cells on scaffold could promote new bone formation in nude mice and enhance the osteointegration for dental implants in canine, which confirmed their osteogenic capacity, and showed that cells derived from cryopreserved bone were comparable to that of fresh bone in terms of the ability to promote osteogenesis in vivo. This work demonstrates that cryopreserved bone grafts may represent a novel, accessible source of cells for tissue regeneration therapy, and the results of our study may also stimulate the development of other cryopreservation techniques in basic and clinical studies.     

Characterization of human hepatocytes isolated from non-transplantable livers

INTRODUCTION: The successful use of hepatocytes depends on a reliable demonstration of the functional and morphological integrity of isolated cells. Herein we investigated whether the isolation and cryopreservation of primary human hepatocytes can compromise cell viability and liver-specific characteristics. MATERIAL/METHODS: Hepatocytes were isolated from encapsulated human liver segments by a modified 2-step perfusion technique. Isolated cells were Percoll-purified, cryopreserved, and stored in liquid nitrogen for 1-12 months. For rapid assessment of fresh and cryopreserve/thawed hepatocyte yield and viability, the cells were stained with trypan blue or labeled with fluorochromes. For immunocytochemical analysis, the cells were labeled with monoclonal antibodies for the presence of the following antigens and chemokines: CD3, CD45Ro, CD45Ra, CD34, CD68, CD90, CD95, CD20, HLA-DR, Ki67, PCNA, Bcl-2, p53, CXCR3, CXCR4, and SDF-1. The cells were tested for several specific functions, such as ureagenesis, energy status, MTT activity, lactate dehydrogenase leakage, and total CYP450 content. RESULTS: Assessment of both freshly isolated (Percoll-purified) and cryopreserved/thawed hepatocytes revealed a low constitutive level of contamination by non-parenchymal cells compared with crude (unpurified) preparations and tissue sections. All viable hepatocytes showed intact morphology and retained CYP450 protein, energy status, and urea synthesis. CONCLUSIONS: Modifications in hepatocyte preparations, such as depletion of dead, damaged, and nonparenchymal cells, improves cell purity, which can be adapted to further evaluation of hepatocyte immunogenicity. These data illustrate the importance and feasibility of human hepatocyte banking.     

Magnetic cryopreservation for dental pulp stem cells

Magnetic cryopreservation has been successfully used for tooth banking with satisfactory implantation outcomes, suggesting that the method preserves human periodontal ligament cells and dental pulp stem cells (DPSCs). Therefore, magnetic cryopreservation may be applied for the preservation of DPSCs; however, this method has not been evaluated yet. A reliable cryopreservation method for live-cell preservation is important for the clinical applications of regenerative medicine. The conventional slow-freezing procedure with 10% dimethylsulfoxide (DMSO) may not be appropriate for stem cell-based therapies because DMSO is cytotoxic. The objective of this study was to investigate whether magnetic cryopreservation can be applied for DPSC cryopreservation. Cells isolated from human dental pulp were subjected to magnetic cryopreservation. Postthawing cell viability, adhesion, proliferation, expression of markers for mesenchymal stem cells (MSCs), differentiation ability of magnetically cryopreserved DPSCs and DNA stability were compared to those of cells subjected to the conventional slow-freezing method. The results indicated that a serum-free cryopreservation medium (SFM) containing 3% DMSO is optimal for magnetic cryopreservation. Post-thaw magnetically cryopreserved DPSCs express MSC markers, and perform osteogenesis and adipogenesis after induction similarly to fresh MSCs. No significant DNA damage was found in magnetically cryopreserved DPSCs. Magnetic cryopreservation is thus a reliable and effective method for storage of DPSCs. The smaller amount of DMSO required in SFM for cryopreservation is beneficial for the clinical applications of post-thaw cells in regenerative medicine.