Endometrial secretory proteins enhance early embryo development

Purpose To evaluate the role of endometrial stromal cells and their secretory proteins in early embryo development, two-celled CB6F1 mouse embryos were cultured alone or cocultured with human endometrial stromal cells in various culture conditions.Results The percentage of embryo blastocyst formation, hatching, and outgrowth was significantly greater in (1) coculture with endometrial stromal cells than in a cell-free control when both coculture and control were carried out in protein-free medium or in RPMI 1640 plus 10% fetal calf serum; (2) coculture with hormone (i.e., progesterone plus relaxin)-treated cells than in coculture with hormone-nontreated cells; and (3) media supplemented with isolated endometrial secretory proteins than in media supplemented with BSA (0.35%). Embryo development was not found to be significantly different in coculture and in media supplemented with endometrial secretory protein.Conclusion Our data provides credence to the theory that endometrial stromal cells enhance embryo development by secreting specific proteins that are beneficial to embryo growth in vitro.Presented at the 48th Annual Meeting of the American Fertility Society, New Orleans, Louisiana, October 31–November 5, 1992.     

Coculture of mouse embryos with cryopreserved human oviduct epithelial cells

Purpose The effect of coculturing mouse embryos with cryopreserved human oviduct epithelial cells was investigated. The cryopreserved cells in Cellbanker were thawed and cultured in Richard D. Goldsby culture medium to establish monolayers. Two-cell-stage mouse embryos were cultured alone (control group) or cocultured with monolayers established from cryopreserved cells (cryopreserved coculture group) or from fresh cells (fresh coculture group). The rates of embryo development and the qualities of the blastocysts in the three groups were compared.Results The two coculture groups had significantly higher blastocyst development rates (cryopreserved coculture group, 81.6%; fresh coculture group, 82.2%) than the control group (63.1%). The two coculture groups had significantly more blastomeres (cryopreserved coculture group, 108.3 ± 25.9; fresh coculture group, 108.4 ± 25.1) than the control group (87.7 ± 31.9).Conclusion The method of cryopreservation of human oviduct epithelial cells using Cellbanker is simpler than conventional cryopreservation methods. These cryopreserved human oviduct epithelial cells may provide a constant supply of cells for coculture for in vitro fertilization and embryo transfer.     

Improvement in blastocyst hatching of mouse embryos cocultured with human placental cells

Purpose Although the hatching of embryos is an important phenomenon, the mechanism of hatching remains controversial. Therefore, we attempted to develop a new coculture system with human placental cells to investigate the hatching of mouse embryos.Results In our new system there was no difference in development from the two-cell stage to blastocysts between embryos cultured with a T6 medium and embryos cocultured with human placental cells at 1 × 10⁵, 5 × 10⁵, and 1 × 10⁶ cells/ml. However, the hatching-rate cell number increased significantly in embryos cocultured with placental cells compared to embryos cultured without placental cells. [³H]Thymidine uptake did not show any significant difference from the beginning of in vitro culture to the hatching stage between the coculture group and the control group. Nevertheless, the [³H]uridine uptake was significantly different in the two groups, measuring 2167 ± 532 cpm/10 embryos in the coculture group and 804 ± 86 cpm/10 embryos in the control group at 114 hr after human chorionic gonadotropin injection (P < 0.01).Conclusion These results therefore seem to indicate that the hatching of blastocysts depends on the protein synthesis of the embryos and not on DNA duplications.     

14C放射性核素标记硫脲嘧啶掺入法测定黑素小体转移

目的 建立用14C放射性核素标记的硫脲嘧啶（14C-TU）掺入定量测定黑素小体转移的方法。方法 14C-TU被黑素细胞摄入后能迅速与黑素生成中间产物特异性地结合而掺入新生黑素中，用经14C-TU预标记过夜的melan-a小鼠黑素细胞（MC）与SP-1小鼠角质形成细胞（KC）建立共培养体系，并在共培养后的不同时间，采用二次差异胰酶消化法再将两种细胞分离，用液体闪烁计数仪测定KC内被转移的放射活性量以示黑素小体转移量。同时，应用此方法初步观察毛喉素（一种PKA激动剂）和烟酰胺对黑素小体转移的影响。结果 ①相同数目（2.5 × 105）的MC和KC分别用1 μCi 14C-TU标记12 h或48 h，放射活性测定显示，MC的放射活性（c/min）分别是KC的80或66倍，表明14C-TU能高度特异地标记新生黑素，是一理想示踪黑素小体转移的标记物。②与未处理对照组相比，20 μmol/L毛喉素能增加黑素小体转移近2.3倍，而1 g/L烟酰胺能抑制黑素小体转移达0.67倍。③MC与KC共培养8 ~ 12 h，MC树突伸展良好，并已有黑素小体发生转移，此时尚未观察到毛喉素诱导的MC增殖。二次差异胰酶消化法用于共培养体系中的KC分离能获得满意的效果（KC纯度约为84.5%）。结论 建立体外的MC和KC共培养结合14C-TU掺入法能定量观察毛喉素和烟酰胺对黑素小体转移的影响。     

大鼠肝细胞与Kupffer细胞共同培养对两者功能和形态影响

目的观察大鼠肝实质细胞与Kupffer细胞体外共同培养时对两者生长、形态及功能的影响。方法采用原位二步Ⅳ型胶原灌注法、Percoll液密度梯度离心法分离Wistar大鼠肝实质细胞与Kupffer细胞;体外将肝实质细胞与Kupffer细胞按6∶1比例共同培养。观察不同情况下肝细胞生存时间和形态,每隔24 h检测培养上清液中清蛋白和谷丙转氨酶(ALT)、谷草转氨酶(AST)的水平,并在培养36 h用放免法检测上清液中白细胞介素1(IL-1)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)含量。结果单独培养组肝细胞的生长、增殖迅速,并向正常肝细胞的形态演变,肝细胞可培养存活至15 d;共同培养组肝细胞生长增殖缓慢,细胞可培养存活至10 d。共同培养组上清液中清蛋白水平在24、36、48、60 h比单独培养组低(t=2.551~3.139,P<0.05);ALT、AST水平在24、36、48、60 h比单独培养组高(t=2.446~3.108,P<0.05);共同培养组Kupffer细胞保持IL-1I、L-6、TNF-α分泌功能,而单独培养组未检测到IL-1I、L-6、TNF-α。结论大鼠Kupffer细胞和肝实质细胞在适宜的培养条件下可进行共同培养,二者可以保持良好的分泌功能,可用于实验研究。     

复合培养对低氧时肺动脉平滑肌细胞表达细胞因子的调节

目的 研究低氧对肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)白细胞介素-1β、4、10、13(IL-1β、4、10、13)活性的影响,及复合培养下肺微血管内皮细胞(pulmonary microvascular endothelial cells,PMVECs)对其调控作用.方法 大鼠PASMCs单独培养或与PMVECs复合培养,分为正常组、低氧作用2 h(H2)、6h(H6)、12 h(H12)、24 h(H24)组,采用ELISA法测定各组培养细胞上清中IL-1β、IL-4、IL-10、IL-13的活性.结果 低氧刺激后PASMCs的IL-1β、IL-4、IL-10、IL-13活性2 h开始升高,6 h达高峰,12 h降低;各时相点复合培养组均低于对应的单独培养组(P＜0.01).结论 低氧可以激活PASMCs的IL-113、IL-4、IL-10、IL-13活性,在复合培养条件下,PMVECs对此效应具有下调性影响.     

Myelinating cocultures of rodent stem cell line‐derived neurons and immortalized Schwann cells

Myelination is one of the most remarkable biological events in the neuron–glia interactions for the development of the mammalian nervous system. To elucidate molecular mechanisms of cell‐to‐cell interactions in myelin synthesis in vitro, establishment of the myelinating system in cocultures of continuous neuronal and glial cell lines are desirable. In the present study, we performed co‐culture experiments using rat neural stem cell‐derived neurons or mouse embryonic stem (ES) cell‐derived motoneurons with immortalized rat IFRS1 Schwann cells to establish myelinating cultures between these cell lines. Differentiated neurons derived from an adult rat neural stem cell line 1464R or motoneurons derived from a mouse ES cell line NCH4.3, were mixed with IFRS1 Schwann cells, plated, and maintained in serum‐free F12 medium with B27 supplement, ascorbic acid, and glial cell line‐derived neurotrophic factor. Myelin formation was demonstrated by electron microscopy at 4 weeks in cocultures of 1464R‐derived neurons or NCH4.3‐derived motoneurons with IFRS1 Schwann cells. These in vitro coculture systems utilizing the rodent stable stem and Schwann cell lines can be useful in studies of peripheral nerve development and regeneration.     

A Comparison of Methods for Isolation of Limbal Niche Cells: Maintenance of Limbal Epithelial Stem/Progenitor Cells

PurposeLimbal niche cells (LNCs) play a vital role in the maintenance of limbal epithelial stem/progenitor cells (LESCs). Four methods have been reported to isolate and expand LNCs: digestion by collagenase alone (C-LNC), collagenase following dispase removal of the limbal epithelium (DC-LNC), dissection of dispase-isolated limbal epithelial sheets (D-LNC), and explant cultures of limbal stromal tissues (Ex-LNC). This study aimed to isolate LNCs using those four methods and to compare their capacity to maintain LESCs.MethodsLNCs were isolated from the rat corneal limbus by the following methods: C-LNC, DC-LNC, D-LNC, and Ex-LNC. Quantitative real-time PCR and immunofluorescence staining were used to analyze the expression of embryonic stem cell (ESC) markers. The ability to maintain LESCs was assessed on the basis of colony-forming capacity and the expression of progenitor, proliferation, and differentiation markers in three-dimensional (3D) Matrigel and Transwell systems. Notch signaling of LESCs supported by different LNCs in Transwell inserts was analyzed by quantitative real-time PCR.ResultsDC-LNCs exhibited lower expression of CK12 during isolation and expansion. Among P4-expanded LNCs, DC-LNCs expressed significantly higher levels of Sox2, Oct4, Nanog, and N-cadherin than C-LNCs, D-LNCs, and Ex-LNCs. Compared with other LNCs, DC-LNCs were more effective in maintaining LESCs with higher holoclone-forming efficiency, greater expression of ΔNp63α and Ki67, and lower expression of CK12. DC-LNCs were also more capable of downregulating Notch signaling of LESCs.ConclusionsDC-LNCs were more effective in expressing ESC markers and maintaining LESCs compared to other LNCs. This study identifies an optimal method for the isolation of LNCs in tissue engineering and ocular surface reconstruction.     

多细胞球样体的制备及其在肿瘤治疗研究中的应用

多细胞球样体(MCS)模型具有与体内细胞相似的生理或病理特性,能够更好地模拟细胞在体内的生物学行为,被广泛应用于治疗效能检测中,包括放疗、化疗、免疫治疗、联合治疗等;基于MCS建立的共培养系统为研究同种或异种细胞间的关系提供了良好的平台。本文将对多细胞球样体的制备及应用尤其是近年的新进展做一简要综述。