Cloning of human heterohybridoma cell lines using chronic lymphocytic leukemia B cells as a feeder layer.

B cells from the peripheral blood of chronic lymphocytic leukemia patients were isolated by gradient density centrifugation and used without irradiation as a feeder layer in the cloning of human heterohybridoma cell lines by limiting dilution. Cloning efficiencies were high with all the cell lines tested. These feeder leukemia B cells could also be successfully used after having been stored in liquid nitrogen.     

Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells

Derivation and culture of human embryonic stem cells (hESCs) without animal-derived material would be optimal for cell transplantation. We derived two new hES (HS293 and HS306) and 10 early cell lines using serum replacement (SR) medium instead of conventional fetal calf serum and human foreskin fibroblasts as feeder cells. Line HS293 has been in continuous culture, with a passage time of 5-8 days, since October 2003 and is at passage level 56. Line HS306 has been cultured since February 2004, now at passage 41. The lines express markers of pluripotent hESCs (Oct-4, SSEA-4, TRA-1-60, TRA-1-81, GCTM-2, and alkaline phosphatase). The pluripotency has been shown in embryoid bodies in vitro, and the pluripotency of line 293 has also been shown in vivo by teratoma formation in severe combined immunodeficiency/beige mice. The karyotype of HS293 is 46,XY, and that of HS306 is 46,XX. Ten more early lines have been derived under similar conditions since September 2004. We conclude that hESC lines can be successfully derived using SR medium and postnatal human fibroblasts as feeder cells. This is a step toward xeno-free conditions and facilitates the use of these cells in transplantation.     

Transgenic human fetal fibroblasts as feeder layer for human embryonic stem cell lineage selection

Successful gene targeting in human embryonic stem (hES) cells requires the use of primary fibroblast feeder layers, which assist in the maintenance of the pluripotent state of hES cells. Such feeder layers must also survive any further selection strategy for hES cells. Here we report the production of a novel transgenic human fetal fibroblast (tHFF) as a feeder layer that is resistant to puromycin and can be used for gene targeting and selection of positive clones in hES cells. tHFFs survive under a wide range of puromycin concentrations (0.5-2 microg/ml) and also supports the undifferentiated growth of hES cells. We have demonstrated here that tHFFs are suitable for selecting Envy-hES cells that were transfected with a green fluorescent protein-small interfering RNA (GFP-siRNA) plasmid construct to induce GFP gene down-regulation. The later studies were designed to isolate and propagate stably knockdown cells. tHFFs thus can be used for targeting other genes that would serve as a model to select and understand the differentiation process in hES cells.     

Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers

BACKGROUND: Recently, human embryonic stem (hES) cells have become very important resources for basic research on cell replacement therapy and other medical applications. The purpose of this study was to test whether pluripotent hES cell lines could be successfully derived from frozen-thawed embryos that were destined to be discarded after 5 years in a routine human IVF-embryo transfer programme and whether an STO cell feeder layer can be used for the culture of hES cells. METHODS: Donated frozen embryos (blastocysts or pronuclear) were thawed, and recovered or in vitro developed blastocysts were immunosurgically treated. All inner cell masses were cultured continuously on an STO cell feeder layer and then presumed hES cell colonies were characterized. RESULTS: Seven and two cell lines were established from frozen-thawed blastocysts (7/20, 35.0%) and pronuclear stage embryos (2/20, 10.0%), respectively. The doubling time of hES cells on the immortal STO cell feeder layer was approximately 36 h, similar to that of cells grown using fresh mouse embryonic fibroblast (MEF) feeder conditions. Subcultured hES cell colonies showed strong positive immunostaining for alkaline phosphatase, stage-specific embryonic antigen-4 (SSEA-4) and tumour rejection antigen 1-60 (TRA1-60) cell surface markers. Also, the hES colonies retained normal karyotypes and Oct-4 expression in prolonged subculture. When in vitro differentiation of hES cells was induced by retinoic acid, three embryonic germ layer cells were identified by RT-PCR or indirect immunocytochemistry. CONCLUSIONS: This study indicates that establishment of hES cells from frozen-thawed blastocysts minimizes the ethical problem associated with the use of human embryos in research and that the STO cell feeder layer can be used for the culture of hES cells.     

Isolation and characterization of human multiple myeloma cell enriched populations

We developed a simple and rapid method to enrich tumor cells within bone marrow (BM) aspirates from patients with multiple myeloma (MM). Thirty patients with a median of 50% (8-85%) MM cells by morphology and 55% (6--85%) MM cells identified by CD38+CD45-cell surface phenotype were studied. BM mononuclear cells (BMMCs) were isolated by Ficoll Hypaque sedimentation and incubated with a cocktail of mouse monoclonal antibodies (mAbs) directed against CD3 (T cells); CD11b and CD14 (monocytes); CD33 (myeloid cells), CD45 and CD45RA (leucocyte common antigen); CD32 as well as glycophorin A. After the addition of anti-mouse Fc Ig-coated immunomagnetic beads, mAb-bound cells were removed in a magnetic field. The residual cell populations were enriched for MM cells, evidenced by >95% plasma cell morphology and >95% CD38+CD45RA-cell surface phenotype. Since this method requires only two short incubations, cell losses were minimal and the yield of MM cells was therefore high (>95%). Viability of the MM-cell enriched fractions was 99%, and these cells were functional in assays of proliferation, cell cycle analysis and immunoglobulin secretion. This immunomagnetic bead depletion method therefore permits the ready isolation of homogeneous populations of patient MM cells for use in both cellular and molecular studies.     

Production of hybridoma growth factor by human monocytes

Human mononuclear leukocytes produce a growth factor (HGF) for hybridoma and plasmacytoma cells. HGF has recently been proven to be identical to IFN-beta 2, 26-kDa protein and BSF-2. HGF can be quantitated in a proliferation assay with the HGF-dependent hybridoma cell line B13.29. By selection of an extremely sensitive variant of this cell line, we were able to measure HGF production of single cells. Limiting dilution analysis of the producing cells in combination with size, density and adherence characteristics showed that HGF is produced by monocytes and not by lymphocytes. There was no need for the monocytes to be stimulated but the cells did require the presence of serum. This serum requirement could be met by purified bovine serum albumin, but not by other proteins like ovalbumin or human gamma-globulin. HGF production in vitro by monocytes starts after 2 h of incubation and is completed within 24 h.     

Fabrication of transplantable human oral mucosal epithelial cell sheets using temperature-responsive culture inserts without feeder layer cells

To exclude bacteria- or animal-derived factors from cultured fabrication of transplantable epithelial cell sheets, primary human oral mucosal epithelial cells were seeded on temperature-responsive culture inserts having submicron-scale pores. Supplying culture medium containing human autologous serum to both apical and basal sides of human epithelial cells allows these cells to grow to confluence. These proliferating cells created stratified epithelial layers even when 3T3 feeder layers and fetal bovine serum were eliminated from culture. Normal keratin expression profiles were obtained with these cells, and basal and midlayer cells expressed p63, a putative stem/progenitor marker. These results suggest that temperature-responsive culture inserts can be useful in clinical settings that require the exclusion of xenogeneic factors.     

The selection of somatic cell hybrids between human lymphoma cell lines

Somatic cell hybrids have been selected between three pairs of established human lymphoid cell lines producing pure lines of proliferating hybrid cells: Raji/Namalwa, Raji/Daudi, and Raji/BJAB. The hybrid cell lines have been characterized with respect to isozyme pattern, volume, and karyotype.Paper 1 of the series, Somatic cell hybrids between human lymphoma cell lines.     

成纤维细胞饲养层饲养人角朊细胞的机制研究

目的 探讨人成纤维细胞(HFB)是否可作为饲养层支持人角朊细胞(KC)的生长.方法 分离培养人真皮FB,用丝裂霉素C处理制成饲养层,应用RT-PCR、免疫细胞化学染色方法检测Ⅰ型、Ⅲ型前胶原mRNA和胶原蛋白的表达;在FB饲养层上接种人KC,观察其促进KC贴壁、生长、移行及分化的作用;并设置NIH3T3细胞饲养层为对照组,在其上接种人KC,观察KC贴壁数和膜片完全融合时间.结果 FB饲养层Ⅰ型前胶原mRNA的表达有所增加(P＜0.05)、Ⅲ型前胶原mRNA的表达无明显变化,抗Ⅰ型、Ⅲ型胶原蛋白抗体阳性;以适宜密度接种的成纤维细胞饲养层,KC贴壁、生长和分化良好;二种饲养层KC贴壁数和膜片完全融合时间无差异.结论 人真皮FB饲养层能够分泌Ⅰ型和Ⅲ型胶原,促进人KC生长并形成复层细胞.     

Anti-proliferative effect of levamisole on human myeloma cell lines in vitro

Levamisole has been employed as an immunomodulatory agent in conjunction with 5-fluorouracil in the treatment of colon cancer relapse. At high doses, levamisole has been shown to have both anti-cancer and immunosuppressive activities. In vitro, levamisole has been shown to potentiate the anti-proliferative effect of 5-fluorouracil in several types of tumor cell lines; however, its mechanism of cytotoxic action and its molecular targets in cells remains to be elucidated. Here, the effect of levamisole on the proliferative response of the human multiple myeloma cell lines RPMI 8226 and U266B1 was studied in vitro. Treatment of both lines with varying concentrations of levamisole for 48 and 72?h in culture resulted in a significant inhibition of proliferation (unstimulated) in a dose-dependent manner, as assessed by an 3-[(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide dye assay. Furthermore, measurements of cell viability (using a trypan blue dye exclusion assay) clearly showed that the levamisole was cytotoxic. The preliminary evaluation of the mechanism of this cytotoxic effect revealed that this drug induced apoptosis in the myeloma cells, as evidenced by increases in the levels of DNA fragmentation, release of cytochrome c into the cytoplasm, and the activation of caspase-3 activity in the cells. The results of these studies strongly suggest that levamisole could be a potent anti-myeloma agent and might be considered in the treatment of multiple myeloma in the future.     

包皮成纤维细胞作为饲养层对人胚胎生殖细胞生长的影响

目的 以人包皮成纤维细胞为饲养层体外培养人胚胎生殖细胞,观察饲养层对其生长的影响,并对人胚胎生殖细胞进行鉴定。 方法 分离培养4～5岁小儿包皮成纤维细胞,取P₃～P₃₀代细胞用丝裂霉素灭活后铺板备用,酶联免疫吸附测定（ELISA）双抗夹心法检测其分泌物成纤维细胞生长因子（FGF）的含量;分离培养5～11周人胚胎原始生殖细胞,在不添加任何细胞因子的人包皮成纤维细胞饲养层上培养人胚胎生殖细胞;细胞化学法检测人胚胎生殖细胞碱性磷酸酶的活性,免疫细胞化学法检测其胚胎表面特异性抗原SSEA-1及SSEA-4的表达,RT-PCR法检测Oct-4的表达。 结果 包皮成纤维细胞可以传60代以上,P₃～P₃₀代均适宜作饲养层;P₃～P₃₀代包皮成纤维细胞上清液中FGF含量在(172.09±2.66)pg/L～(245.25±1.66)pg/L之间波动。分离培养的人胚胎生殖细胞在饲养层上可形成典型的胚胎生殖细胞集落,体外连续培养超过3代,其碱性磷酸酶活性呈强阳性,集落未分化标志检测显示SSEA-l、SSEA-4呈阳性,Oct-4表达阳性。 结论 用人包皮成纤维细胞作为饲养层能支持人胚胎生殖细胞的生长并维持未分化状态。     

人胚胎成纤维细胞饲养层的制备

背景：极小胚胎样干细胞是近年来发现的一种具有类似胚胎干细胞生物学特性的非造血干细胞,但对其体外培养扩增的方法报道极少。有研究推测,人胚胎成纤维细胞能为人骨髓极小胚胎样干细胞体外培养扩增提供良好的微环境。 目的：从人胚胎躯干中分离、培养人胚胎成纤维细胞,制备人胚胎成纤维细胞饲养层用于人骨髓极小胚胎样干细胞的培养。 方法：利用胰酶消化法从孕5-9周龄人胚胎躯干中分离培养人胚胎成纤维细胞。制作饲养层,使用不同浓度丝裂霉素C处理后,用于培养分选后的人骨髓极小胚胎样干细胞,以细胞形态、生长曲线作为胚胎成纤维细胞和饲养层的评价指标。 结果与结论：从人胚胎中成功分离培养出人胚胎成纤维细胞,该细胞可传代24代以上,且经过传代及冻存复苏后生物学特性无改变。丝裂酶素C低于12 mg/L时,人胚胎成纤维细胞增殖不能完全抑制;高于14 mg/L,人胚胎成纤维细胞可能死亡。12 mg/L丝裂霉素C作用3 h后能较好地抑制人胚胎成纤维细胞的增殖,并且保持其活力约2周,可以在很长一段时间内用作人骨髓极小胚胎样干细胞的饲养层。     

抗人成骨肉瘤杂交瘤细胞系的建立及其特性鉴定

目的 建立分泌抗人成骨肉瘤单克隆抗体（mAb)的杂交瘤细胞系，并对其分泌的mAb进行鉴定。方法 用成骨肉瘤细胞系（OSPS-9607）免疫Balb/c小鼠，取脾细胞按常规方法融合，筛选，克隆化及腹水mAb制备，取骨肉瘤手术标本（60例）及正常组织（6种），用mAb进行免疫组化ABC染色。结果 筛选出2株能持续，稳定分泌特异性抗骨肉瘤mAb的杂交瘤细胞株（6E5和3F7），2株杂交瘤细胞经过100d连续培养，分泌mAb的特性稳定，mAb特异性强，效价高。免疫组化染色检测表明，mAb6E5和3F7针对的抗原，在成骨肉瘤组织及成骨肉瘤细胞系均呈高表达（70%）。针对mAb6E5的抗原主要分布于细胞核上，针对mAb3F7的抗原主要分布于细胞浆内，6种正常组织中除胃粘膜可见弱阳性反应外，其余未见明确的阳性反应。结论 此两株杂交瘤细胞分泌的mAb对成骨肉瘤细胞具有特异亲和性，为成骨肉瘤的早期诊断。免疫治疗及发现成骨肉瘤新的标志物奠定了基础。     

Characterization of anti-reovirus immunoglobulins secreted by cloned hybridoma cell lines

Nineteen hybridoma cell lines derived from the spleen cells of a mouse immunized with reovirus serotype 3 (strain Dearing) were cloned and the IgGs secreted by them characterized. Four of the IgGs were directed against polypeptide μNS and two against polypeptide σNS, the two nonstructural reovirus-specified polypeptides. Two IgGs were directed against polypeptide λ2 and two against polypeptide σ2, both components of the reovirus inner capsid shell. Four IgGs were directed against polypeptide σ1, the most type specific of all reovirus-specified polypeptides, three against polypeptide μl/gmlC and one against polypeptide σ3, all of them components of the reovirus outer capsid shell. One IgG was directed against a complex composed of polypeptides μl/gmlC and σ3, but failed to react with either of these polypeptides alone. Thus the 19 hybridoma cell lines secreted IgGs with specificities for 7 of the 10 reovirus-specified proteins. The ability of all these IgGs to precipitate the proteins specified by the two heterologous reovirus serotypes, serotypes 1 and 2, was also measured.     

抗伏马菌素B_1单克隆抗体的制备及鉴定

目的建立抗伏马菌素B1（FB1）的单克隆杂交瘤细胞株,制备抗FB1的单克隆抗体（McAb）。方法采用FB1-KLH偶联物小剂量长周期免疫BALB/c小鼠后,采用细胞融合方法获得分泌抗FB1的杂交瘤细胞株,采用多次亚克隆的方法建立了4株稳定分泌抗FB1抗体的杂交瘤细胞株。腹水诱生法获得大量McAb,采用抗体亚类测定试剂盒鉴定抗体的亚类,SDS-PAGE测定抗体分子量,McAb的特异性和灵敏度用间接竞争抑制ELISA。结果免疫小鼠血清检测结果显示经过5次免疫后血清的效价稳定在1×10-6,经过4次亚克隆后建立4株稳定分泌抗FB1抗体的杂交瘤细胞株,获得腹水,纯化抗体,抗体亚类属于IgG2a类,轻链为κ,轻链和重链的相对分子质量分别是55000和32000,ELISA检测抗体特异性显示可与FB1发生特异性反应,采用此抗体建立间接竞争抑制ELISA方法的线性范围在2～500ng/ml。结论制备了特异性和灵敏度较高的抗FB1单克隆抗体,为建立伏马菌素免疫学检测方法打下良好基础。     

5种不同人源性饲养层对人胚胎干细胞生长支持情况的研究

目的比较5种不同人源性饲养层对人胚胎干细胞生长支持的情况。方法经签署相关知情同意书后,按人源性饲养层的标准化操作规范获得包皮、减胎、胎儿皮肤、输卵管、睾丸组织的成纤维细胞,经抗波形白荧光染色鉴定,扩增培养后传代并冻存,经丝C裂霉素处理,将胚胎干细胞NS-1接种于其上,观察NS-1贴壁生长情况,AKP染色计算NS-1分化率。结果获得了5种人源性饲养层,分别为包皮、减胎、输卵管、胎儿皮肤,睾丸组织的成纤维细胞,经抗波形蛋白鉴定,阳性表达,抗角型蛋白呈阴性。5种人源性饲养层在hESCs的贴壁率及生长率上与MEF均无统计学差异,但在长期的培养过程中,MEF支持的hESCs未分化率明显高于5种人源性饲养层支持的hESCs未分化率。结论在常规的培养系统中,5种人源性饲养层不能长期支持人胚胎干细胞的生长。     

Parvoviruses as contaminants of permanent human cell lines

Summary Tissue culture harvests of KBSH-virus were shown to contain two types of particles differing in their nucleic acid contents and banding in CsCl at 1.395 g/ml and 1.35 g/ml, respectively. The DNA's of these particles proved to be of single-stranded configuration.Native and alkali-denatured DNA extracted from virus particles with a mean density of 1.395 g/ml sedimented at around 24 S in neutral solution and at 18.3 S in alkaline solution and banded at 1.724 g/ml in neutral CsCl.This figures correspond to a molecular weight of 1.4 × 10⁶ or 26.5% of a total particle weight of 5.3 × 10⁶. After denaturation as well as after storage of native DNA two additional single-stranded nucleic acid species appear sedimenting at 19–20 S and at about 16 S.The DNA extracted from particles banding at 1.35 g/ml lacks homogeneity; the majority of the DNA molecules, however, sedimented at about 10 S consistent with a molecular weight of 0.27 × 10⁶,i.e. only 20% of complete KBSH-DNA.Part of the results of this paper have been already presented at the 3. Arbeitstagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie, 8.–10. October 1970 in Mainz.     

Fusion between immunoglobulin-secreting and nonsecreting myeloma cell lines

The defects in two nonsecreting variant clones of the mouse plasmacytoma MOPC 21 (P3) were studied by tissue culture methods. The variants (NSI and NSIII) do not synthesize detectable heavy chains. NSI synthesizes, but does not secrete, light chains and NSIII does not synthesize light chain. A screening procedure was used allowing the detection of revertant cells secreting immunoglobulin. The method is based on a hemolytic plaque assay using anti-immunoglobulin-coated red cells. No revertants were detected among 2 × 10⁷ cells. Both variant lines were fused to another myeloma line (PI) which secretes a complete immunoglobulin and excess light chains. Analysis of the products by isoelectric focusing showed that in the hybrids there was no reactivation of synthesis of the nonexpressed chains. The defects leading to loss of synthesis cannot therefore be complemented in the hybrid lines. The secretion of light chain in NSI, on the other hand, could be complemented in the hybrid but the light chain was only secreted as part of a new immunoglobulin hybrid molecule.