Although Cell-in-cell structures (CICs) had been documented in human tumors for decades, it is unclear what types of CICs were formed largely due to low resolution of traditional way such as H&E staining. In this work, we employed immunofluorescent method to stain a panel of human tumor samples simultaneously with antibodies against E-cadherin for Epithelium, CD68 for Macrophage and CD45 for Leukocytes, which we termed as “EML method” based on the cells detected. Detail analysis revealed four types of CICs, with tumor cells or macrophage engulfing tumor cells or leukocytes respectively. Interestingly, tumor cells seem to be dominant over macrophage (93% vs 7%) as the engulfer cells in all CICs detected, whereas the overall amount of internalized tumor cells is comparable to that of internalized CD45+ leukocytes (57% vs 43%). The CICs profiles vary from tumor to tumor, which may indicate different malignant stages and/or inflammatory conditions. Given the potential impacts different types of CICs might have on tumor growth, we therefore recommend EML analysis of tumor samples to clarify the correlation of CICs subtypes with clinical prognosis in future researches.

BACKGROUND: Extract of injured pancreatic gland contains specific growth factor, which can promote β cell proliferation and insulin secretion, resulting in insulin regeneration and cell differentiation. OBJECTIVE: To investigate effects of the injured rat pancreatic extract on in vitro trans-differentiation of bone marrow mesenchymal stem cells (BMSCs) into insulin-like cells. DESIGN, TIME AND SETTING: A cytology in vitro experiment was performed at the Institute of Biotechnology, Academy of Military Medical Sciences of Chinese PLA from June 2005 to March 2007. MATERIALS: Six clean male Sprague-Dawley rats were obtained from the Animal Experimental Center of Academy of Military Medical Sciences. Activin A, Retinoic Acid and trypsin inhibitor were bought from Sigma, USA. Streptozotocin was purchased from Sigma company. METHODS: Streptozotocin was intraperitoneally infused into rats. Two days later, pancreatic tissues were obtained when blood glucose exceeded 10 mmol/L. Homogenate was prepared in phosphate buffer saline containing pancreatic protein inhibitor. Supernatant was obtained after twice centrifuge, which was injured pancreatic extract. BMSCs were isolated from rats by density gradient centrifugation. The passage cells were randomly divided into 4 groups when 70%-80% passage cells were confluent. BMSCs in the control group were incubated in low-glucose DMEM containing 2% fetal bovine serum for 11 days. BMSCs in the Activin A and Retinoic Acid (AR) group were separately treated with Activin A and Retinoic Acid each for 24 hours, and with basic fibroblast growth factor, nicotinic amide, nicotinic amide+exendin each for 3 days. BMSCs in the Activin A, Retinoic Acid and injured pancreatic extract (AR+E) group were treated with injured pancreatic extract on the basis of former groups. BMSCs in the injured pancreatic extract (E) group were only incubated with injured pancreatic extract for 11 days. MAIN OUTCOME MEASURES: Phenotypes of cells were examined by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Insulin secretion levels were determined by enzyme labeled immunosorbent assay (ELISA). RESULTS: More insulin-positive cells existed in AR and AR+E groups, and insulin-like structure was bigger and more in the AR+E group compared with the AR group. There were a few insulin-positive cells and insulin-like structure in the E group. Insulin-1 mRNA expression existed in the experimental groups. Insulin secretion was detected in supernatant. Insulin secretion levels were greater in the AR+E group compared with the AR group, and secretion levels were the lowest in the E group (P < 0.05). Each index was negative in the control group. CONCLUSION: On the basis of previous study of Activin A and Retinoic Acid and other maturation factors, the injured pancreatic extract can increase the efficiency of BMSCs differentiation into insulin-like cells, resulting in elevating differentiation efficiency.

利用HEK293细胞在悬浮培养中具有聚集成团的体外培养特性,在250ml的Bellco的搅拌培养体系中,以细胞团粒径、细胞粒径、细胞数、细胞活力、葡萄糖比消耗速率 (qglc)、乳酸比生产速率 (qlac) 和乳酸对葡萄糖得率 (Ylac/glc) 为观察指标,考察了HEK293细胞在Ca2+浓度设置为0μmol/L、250μmol/L、500μmol/L、750μmol/L和1000μmol/L的搅拌培养体系中的细胞团形成、细胞生长和代谢.实验发现:培养基中的Ca2+浓度决定着HEK293细胞在悬浮培养中能否形成细胞团并影响着细胞团的粒径分布和细胞团内细胞相互连接的紧密程度;在250～1000μmol/L的Ca2+浓度范围内,HEK293细胞团的平均粒径与培养基中的Ca2+浓度成正比;Ca2+浓度对以细胞团的形式悬浮培养的HEK293细胞的生长和代谢无明显的影响.实验结果提示,Ca2+浓度是调节HEK293细胞团粒径分布、维持悬浮细胞团中HEK293细胞正常生长和代谢的有效控制参数.

Cells of the human embryonic kidney cell line (HEK 293) grown in repeated suspension and perfusion systems were characterized and described. Cell aggregates that formed immediately after the HEK 293 cells were inoculated in stirred vessels in serum-containing Dulbecco’s modified Eagle’s medium (D-MEM)/F-12 medium. The mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 63 to 239 μm after 1 and 8 days of culture in spinner flasks, respectively. No significant differences in cell performance were observed between HEK 293 cell populations grown as suspended aggregates and those grown as anchored monolayers. Replacing the D-MEM/F-12 with CD 293 medium caused the compact spherical cell aggregates to dissociate into single cells and small irregular aggregates without any apparent effect on cell performance. Moreover, the spherical cell aggregates could reform from individual cells and small aggregates when exposed to the serum-containing D-MEM/F-12 dominant medium. Perfusion culture of HEK 293 cells grown as suspended aggregates in a 7.5-l stirred tank bioreactor for 17 days resulted in a maximum viable cell density of 1.2×107 cells ml−1. These results demonstrate the feasibility and proof-of-concept for using aggregates as an immobilization system in large-scale stirred bioreactors because a small-scale culture can be used as easily as the inoculum for larger bioreactors.