The proliferation and function of human mononuclear leukocytes and natural killer cells in serum-free medium

We recently developed a serum-free (SF) culture medium that supports the growth of several established lymphoid cell lines. In an effort to develop a standardized medium for assay of human natural killer (NK) cell activity, we compared the cytotoxic activity of peripheral blood mononuclear leukocytes (PBL) and purified large granular lymphocytes (LGL) cultured in SF medium containing interleukin 2 (IL-2) or medium containing 10% fetal bovine serum (FBS) plus IL-2. The results indicated that PBL had a 30% increase in cumulative net cell growth and had as high or higher cytotoxic activity after growth in SF medium than in medium containing FBS. Purified LGL had a 50% increase in cumulative net cell growth and persisted approximately 2 weeks longer in culture in medium containing FBS than in SF medium. However, the cytotoxic activity of cells grown in SF medium persisted during the initial 3 weeks of culture. Purified LGL that were maintained and were subcultured at cell densities of 10(6) cells or greater per milliliter of either SF or FBS-containing medium had equivalent levels of cytotoxicity over a 44-day period in either medium compared with cells subcultured at a density of 5 X 10(5) cells per milliliter of medium. NK cells produced a cytotoxic factor (NKCF) in SF medium, and its cytotoxic activity was blocked by 10% FBS. We conclude that the SF medium supplemented with IL-2 can be used as an alternative to FBS-containing medium with IL-2 for the growth of NK cells and is advantageous for the production of NKCF.     

一种适合肝细胞生长的无血清培养基*

背景：寻找一种既能适合细胞生长又避免添加血清的培养基迫在眉睫,无血清培养应运而生。 目的：探索一种适用于C3A细胞生长的无血清培养基,为肝细胞用于临床治疗以及生物人工肝走入临床奠定基础。 方法：分别用无血清培养基、完全培养基和基础培养基培养C3A细胞,观察细胞的生长状态,绘制细胞生长曲线和活力曲线,全自动生化分析仪测定上清中谷丙转氨酶漏出量、尿素含量,放射免疫分析法检测白蛋白分泌量,实时荧光定量PCR检测基因水平的变化情况。 结果与结论：3种培养基培养的细胞生长良好,无血清培养基组与完全培养基组细胞密度在培养周期内,除第7天以外差异无显著性意义(P > 0.05)。培养第4~7天,其他两组明显高于基础培养基组(P < 0.05);无血清培养基尿素合成水平>完全培养基>基础培养基(P < 0.05)。Real-time qPCR显示,无血清培养基组相对于完全培养基组仅CK18、CK19和HNF4α基因表达量下降(P < 0.05)。说明实验研制了一种适合于肝细胞(C3A)生长的无血清培养基,与传统血清培养能达到同样的效果。 关键词：培养基;无血清培养;C3A细胞;肝细胞;生物人工肝;PCR检测 doi:10.3969/j.issn.1673-8225.2012.11.015      

Comparison of Chalquest and Hayflick media, with and without ammonium reineckate, for isolating Mycoplasma pulmonis from rats.

Chalquest and Hayflick media with and without ammonium reineckate were compared for isolation of Mycoplasma pulmonis from the nasopharyngeal ducts, tracheobronchial trees, and middle ears of 66 naturally infected rats. The results show that 92% (366 of 396) of the samples were positive for M. pulmonis in Chalquest medium with and without ammonium reineckate and 66% (260 of 396) were positive in Hayflick medium with and without ammonium reineckate (P less than 0.001). An enhancing effect of ammonium reineckate on M. pulmonis isolation was observed only in Hayflick medium; the isolation rate was 76% (151 of 198) in Hayflick medium with ammonium reineckate as compared with 55% (109 of 198) in Hayflick medium without ammonium reineckate (P less than 0.03). No significant differences in isolation rates were observed between Chalquest medium with and without ammonium reineckate. The mean growth time of M. pulmonis on Chalquest medium was 3.4 days as compared with 5.1 days in Hayflick medium, indicating that M. pulmonis can be detected earlier on Chalquest medium than on Hayflick medium. These data indicate that Chalquest medium is superior to Hayflick medium for M. pulmonis isolation from rats.     

Evidence for increased protease activity secreted from cultured fibroblasts from patients with pseudoxanthoma elasticum.

Abnormal polyanion metabolism in cultured fibroblasts from patients with pseudoxanthoma elasticum (PXE) was studied by incubating normal and PXE fibroblasts in culture medium containing 35SO4 for 96 hr and measuring differences in secreted 35SO4-labelled proteoglycans (35S-PG). PXE medium contained less high molecular weight (MW) 35S-PG and more low MW 35S-labelled molecules than normal medium. Addition of diisopropylfluorophosphate (DFP), a serine protease inhibitor, to the media after the 96 hr incubation resulted in no change in the MW distribution of 35SO4-labelled molecules in normal media. However, DFP treated PXE medium contained significantly more high MW 35S-PG than either untreated PXE medium or DFP treated normal medium. High MW 35S-PG was isolated from PXE fibroblast culture medium and incubated with serum free medium from either normal or PXE fibroblast cultures. There was significantly more degradation of this 35S-PG to low MW 35S-labelled molecules by PXE medium than by normal medium. 32P-DFP, which binds to the active site of serine proteases, was added to serum free medium from normal and PXE cultures. The specific radioactivity of PXE medium was 4 times greater than that of normal medium. These lines of evidence are consistent with the hypothesis that a biochemical defect in cultured PXE fibroblasts is the increased secretion of a serine protease that can degrade sulfated proteoglycans.     

提高成年大鼠神经干细胞单克隆形成率的方法

目的　探索提高神经干细胞单克隆形成率的方法并证实所分离培养的神经干细胞仍具有多分化潜能。　方法　对神经干细胞单克隆方法进行改良 ,即在单克隆培养液中加入 1 2原代克隆培养液。将所得到的单细胞克隆球消化、分离、增殖成大量的神经干细胞球 ,用含血清的DMEM分化培养液促其分化。 14d后 ,分别用神经元和胶质细胞的特异性标记物MAP 2标记神经元、GFAP标记星形胶质细胞和CNP标记少突胶质细胞。　结果平均每块含 1 2原代克隆培养液的 96孔培养板中有 2～ 3只孔可形成克隆球 ,而含纯新鲜培养液的培养板中仅 0 5～ 1 0只孔可形成克隆球。这些单细胞克隆球增殖后得到的大量亚细胞系克隆球分化后分别呈MAP 2、GFAP和CNP免疫荧光阳性。　结论　单细胞克隆实验中加入 1 2原代克隆培养液可提高神经干细胞的单克隆形成率 ,单克隆球增殖后得到的大量亚细胞系克隆球亦具有多分化潜能。     

Inhibitory effect of the Isolator blood culture system on growth of Mycobacterium avium-M. intracellulare in BACTEC 12B bottles.

The examination of 6,938 clinical specimens collected during the period January 1991 through December 1992 suggested that the Isolator blood culture system (Wampole) inhibited growth of Mycobacterium avium-M. intracellulare complex (MAC) in BACTEC 12B medium. Of 162 MAC blood culture isolates, 94% were recovered from Lowenstein-Jensen (LJ) medium, while only 50% were recovered from 12B medium. The time to detection with LJ medium was 18 days, while that with 12B medium was 24 days. In contrast, 62% of the 305 MAC nonblood culture isolates were recovered from the LJ medium, while 87% were found in the 12B medium. The time to detection for these cultures was also reversed, i.e., 28 days for LJ medium versus 15 days for 12B medium. Dilution studies using the lysis-anticoagulant reagent from Isolator tubes demonstrated inhibition of both clinical and American Type Culture Collection strains of MAC, even at low concentrations of lysis-anticoagulant reagent. Washing the Isolator blood sediment prior to inoculating the 12B bottles eliminated any growth inhibition. Clinical and experimental data suggest that the use of the Isolator blood culture tube with the BACTEC 12B medium is contraindicated for mycobacterial blood cultures.     

Comparative evaluation of cytotoxicity of cadmium in rat liver cells cultured in serum-containing medium and commercially available serum-free medium

Cadmium (Cd) is an industrial pollutant and carcinogenic metal. Most in vitro Cd toxicity studies have been carried out in various cell lines cultured in 10% fetal bovine serum (FBS) containing medium. In this report, we compared the toxic effect of Cd (0-300 microM) on cell growth, total RNA, total proteins, and antioxidant enzymes in rat normal liver cells cultured in medium with 10% FBS or commercially available serum-free medium for 4 or 8 hours. With Cd concentration at above 100 microM, the total levels of RNA, protein and cell growth decreased in serum-containing medium, while their levels increased in serum-free medium compared to the controls. The glutathione peroxidase and glutathione reductase levels were lower in serum-free medium than in serum-containing medium, indicating less oxidative stress in cells grown in serum-free medium. These results clearly suggest that Cd showed higher toxicity to liver cells grown in serum-containing medium in comparison to commercially available serum-free medium. It is speculated that albumin and other substances present in commercial serum-free medium chelated Cd and thereby protected these cells against Cd toxicity. Even under in vivo conditions, cadmium enters into various organs after passing through blood which contains serum. Based on these studies, it appears that media containing serum may be ideal for in vivo toxicity correlation studies with animal cells.     

半固体培养基法制备单抗的可行性研究

目的建立稳定的甲基纤维素半固体培养基一步法筛选和克隆化杂交瘤细胞的方法。方法常规细胞融合后,用甲基纤维素半固体培养基重悬细胞沉淀,通过优化甲基纤维素半固体培养基中甲基纤维素、血清及L-谷氨酰胺三种成分的浓度,建立稳定高效的杂交瘤细胞克隆化方法。并进行有限稀释法和甲基纤维素半固体培养基法筛选杂交瘤细胞的对照实验。阳性克隆扩大培养,ELISA检测后冻存,再次复苏后ELISA检测呈阳性为稳定杂交瘤细胞株。结果甲基纤维素三种浓度中,甲基纤维素浓度1．0％和1．2％时获得的杂交瘤细胞团个数约为甲基纤维素浓度1．25％时的2倍。而甲基纤维素浓度1．2％时细胞团固定效果较甲基纤维素浓度1．0％更好。通过对梯度稀释的SP 2／0细胞培养后,选择半固体培养基中使用的胎牛血清浓度（25％）,在半固体培养基中含4mmol／L L-谷氨酰胺比含2mmol／L L-谷氨酰胺得到多近一倍的杂交瘤。得到的5株阳性杂交瘤在扩大培养、冻存复苏后抗体分泌稳定,得到5株稳定阳性杂交瘤细胞株。有限稀释法和甲基纤维素半固体培养基法筛选杂交瘤细胞对照实验显示半固体培养基法可节省一半时间。结论建立了稳定的甲基纤维素半固体培养基一步法筛选和克隆化杂交瘤细胞的方法。该法缩短了克隆化的时间,节省了人力和材料．不需要添加饲养细胞和细胞因子,是一种稳定高效的杂交瘤细胞克隆化方法。     

Differential and selective medium for isolation of Yersinia enterocolitica from stools.

A new differential and selective medium, DYS agar, was developed and evaluated from the isolation of Yersinia enterocolitica. Ther bile salts content of the medium resulted in high selectivity, and inclusion of arabinose, lysine, and arginine rendered Y. Enterocolitica very distinct from Proteus spp., Pseudomonas spp., and other members of the family Enterobacteriaceae. DYS medium was more efficient for the isolation of Y. enterocolitica from experimentally inoculated fecal specimens than MacConkey, deoxycholate-citrate, and salmonella-shigella agars. Although the medium showed selectivity similar to that of another relatively new medium. Y medium (a selective medium for Y. enterocolitica containing sodium oxalate). DYS agar was found to be superior to Y medium in terms of differentiation of Y. enterocolitica from other intestinal organisms. DYS medium is simple to prepare.     

Influence of cultivation media on genetic regulatory patterns in Borrelia burgdorferi

Barbour-Stoenner-Kelly II (BSKII) medium and BSKH medium both are routinely used for the cultivation of Borrelia burgdorferi. However, heretofore there have been no studies to compare how these two media affect gene expression patterns in virulent B. burgdorferi. In the present study, we found that some B. burgdorferi strain 297 genes (e.g., ospA, mlp-7A, mlp-8, p22, and lp6.6) that typically are regulated by temperature or pH displayed their predicted pattern of expression when B. burgdorferi was cultivated in BSKH medium; this was not true when spirochetes were cultivated in conventional BSKII medium. The results suggest that BSKH medium is superior to BSKII medium for gene expression studies with B. burgdorferi.     

无蛋白培养中杂交瘤细胞的生长与单克隆抗体分泌

目的：建立应用无蛋白培养基大量制备单克隆抗体（McAb)的生产方法。方法：本实验以3株猪瘟病毒特异单抗杂交瘤细胞为研究对象，通过与常规的有血清培养法比较，研究了无蛋白培养基中杂交瘤的细胞生长特性、抗体分泌效率、活性和纯度。结果：无蛋白培养基中单抗的产量与效价比有血清培养的单抗高2－4倍，而且单抗的纯度显著提高。结论：无蛋白培养基可完全取代常规的有血清培养基，用于生产高滴度、高纯度单克隆抗体。     

Splitting culture medium by air-jet and rewetting for the assessment of the wettability of cultured epithelial cell surfaces

This study found that the phenomenon of rewetting after squeezing culture medium varied in different culture conditions for rat oral mucosal epithelial cells. When culture medium covering over cultured cells was squeezed by an air-jet application, the motion of squeezed culture medium was able to be observed by using a commercially available movie camera. Squeezed width on cells cultured in keratinocyte culture medium (KCM), which contained with fetal bovine serum, was one-sixth of that in FBS-free KCM. This result corresponded to the mucous layer staining statuses of cultured cells in both cases; positive in KCM and negative in FBS-free medium. Furthermore, the gene expression of mucous glycoprotein MUC4 in KCM was 100 times higher than that in FBS-free medium, and the expression of MUC4 protein only showed on the apical surface of cells cultured in KCM. The relative gene expression levels of MUC1, 13, 15, and 16 in both the normal and FBS-free medium were found to be no more than one-thirtieth of that of MUC4 in KCM. The main factor of the wettability difference between KCM and FBS-free medium was speculated to be the difference of MUC4 expression between both media. This method can be a simple technique for testing not only the surface wettability but also the mucous formation of cultured cells.     

不同培养基培养人脐血间充质干细胞的差异

背景：脐血间充质干细胞为干细胞领域的研究热点,但目前传代及扩增此类细胞尚无简单、有效、完美培养方法。 目的：采用不同的培养基分离培养融合状态的间充质干细胞,以筛选一种较好的体外培养人脐血间充质干细胞的方法。 方法：无菌条件下取正常足月剖宫产新生儿的脐血,随机分为5组：低糖DMEM(Dulbecco改良的Eagle培养基)组、高糖DMEM组、α-DMEM组、低糖DMEM+干细胞因子组、低糖DMEM +骨髓间充质干细胞培养上清组。用淋巴细胞分离液分离脐血的单个核细胞。将脐血单个核细胞接种于含体积分数为10%胎牛血清的上述培养基中,放置于37 ℃、体积分数为5%的CO2培养箱内培养,倒置显微镜观察细胞数量和形态的变化并用流式细胞技术分析细胞的表面抗原。 结果与结论：①各组间充质干细胞培养48 h后贴壁细胞数和细胞存活率的比较：低糖DMEM+干细胞因子组、低糖DMEM +骨髓间充质干细胞培养上清组的贴壁细胞数明显多于低糖DMEM组、高糖DMEM组、α-DMEM组(P < 0.05),细胞存活率亦明显高于低糖DMEM组、高糖DMEM组、α-DMEM组(P < 0.05)。②各组间充质干细胞在不同培养时间下生长状态的比较：培养第3,6,9,12,15,18,21天低糖DMEM+干细胞因子组、低糖DMEM+骨髓间充质干细胞培养上清组细胞增殖的速度均快于低糖DMEM组、高糖DMEM组、α-DMEM组(P < 0.05)。低糖DMEM+干细胞因子组与低糖DMEM +骨髓间充质干细胞培养上清组比较差异无显著性意义。结果可见人脐血间充质干细胞与骨髓间充质干细胞培养上清或干细胞因子共孵育,对脐血间充质干细胞体外分离培养及扩增有支持作用。     

Adrenergic development of neural crest cells grown in a defined medium under a reconstituted basement-membrane-like matrix

The embryonic neural crest of vertebrates is the source of a wide variety of adult cell types. We have demonstrated previously that the presence of a reconstituted basement-membrane-like (RBM) gel overlay can dramatically stimulate the development of adrenergic cells in neural crest cultures grown in a complex medium containing horse serum and chick embryo extract. In the present experiments we have analyzed the differentiation of neural crest cells grown in a defined medium with an RBM gel overlay. We found that the presence of the RBM gel promoted the development of catecholamine-containing (CA+) cells in neural crest cultures grown in defined medium compared to cultures grown in this same medium in the absence of the gel. The number of CA+ cells which developed in cultures grown in defined medium in the presence of the RBM gel overlay was similar to that seen in cultures grown in complex medium in the absence of the RBM gel.     

Neisseria confirmation by an enriched, bicarbonate-containing carbohydrate medium.

A sugar fermentation medium for the confirmation of Neisseria and related species was developed. The medium contained a commercial supplement and a hemoglobin source prepared from lysed sheep erythrocytes. Bicarbonate in the medium substituted for a CO2-supplemented atmosphere. The medium was dispensed into screw-capped tubes. This medium was compared to cystine-Trypticase agar and the modified rapid fermentation test in the confirmation of Neisseria species. Performance of the new medium was equivalent to that of the modified rapid fermentation test, but cystine-Trypticase agar failed to confirm a significant number of clinical isolates of Neisseria gonorrhoeae.     

Effect of swab composition and use of swabs versus swab-containing skim milk-tryptone-glucose-glycerol (STGG) on culture- or PCR-based detection of Streptococcus pneumoniae in simulated and clinical respiratory specimens in STGG transport medium

To detect Streptococcus pneumoniae colonization, the nasopharynx is sampled using a swab placed in skim milk-tryptone-glucose-glycerol (STGG) transport medium, and then the swab specimen or STGG medium is cultured or subjected to PCR. We evaluated the effect of swab composition and compared the sensitivities of detection of culture and PCR using swabs and swab-containing medium. Calcium alginate, Dacron polyester, or rayon-tipped swabs were inoculated with pneumococci or were immersed in nasal wash specimens from children and then placed in STGG medium. Swabs and medium inoculated with pneumococci were cultured. Swabs grew significantly more colonies than medium. The number of colonies cultured from rayon swabs or medium was significantly higher than the number cultured from the calcium alginate swab or medium. The number of colonies from both the Dacron polyester swabs and medium were significantly lower than with either calcium alginate or rayon swabs. When DNA was separately extracted from the calcium alginate swab and medium and subjected to PCR for pneumococcal detection from either S. pneumoniae-inoculated swabs or clinical specimens that grew S. pneumoniae, the sensitivity was at least 10 times higher using the swab. With Dacron polyester or rayon-tipped swabs, there was no consistent difference between the sensitivity of PCR using swabs and that of PCR using medium. Thus, calcium alginate swabs may be superior to STGG medium for the culture and PCR-based detection of S. pneumoniae. For culture, rayon swabs are superior and Dacron polyester swabs are inferior. The sensitivity of the swab and swab-containing medium for culture or PCR detection of S. pneumoniae varies with swab composition.     

无血清培养体系原代培养脐带间充质干细胞

背景：以含血清培养基培养的脐带间充质干细胞,存在着进一步应用的障碍。 目的：建立无血清培养体系原代培养脐带间充质干细胞。 方法：取脐带华通氏胶(Wharton’s Jelly),采用机械法将组织胶切碎,1-3 mm3大小,分别培养到含胎牛血清完全培养液中以及无血清培养液中。培养第11,14,17天收获细胞并进行相关检测。在符合2006年制定的干细胞最低评估标准的基础上,以集落形成单元指标评估何种培养体系能获得较多高质量的原代细胞。 结果与结论：无血清培养体系相对于经典的含血清的培养体系而言,细胞生长速度更快。另外,培养第11天,集落形成实验表明无血清培养体系下能获得更多的集落。因此,无血清培养体系可以保持间充质干细胞的特性,为替代含血清培养体系进行脐带间充质干细胞原代培养提供了可能。     

Effect of dipyridamole on adenosine incorporation into hypoxanthine nucleotides of fresh human red cells

Fresh human red cells were incubated for 2 hours in a medium containing adenosine, pyruvate and inorganic phosphate (APP medium), or in APP medium supplemented with 10(-4) M dipyridamole (APPD medium). No measureable amount of ITP was found in fresh red cells, and the average IMP content in these cells was 0.18 +/- 0.09 mumol/g Hb. After 2 hours incubation in APP medium, the IMP content increased almost 8.5-fold to 1.52 +/- 0.78 mumol/g Hb. Under these conditions the ITP level also increased to 1.40 +/- 0.84 mumol/g Hb. After 2 hours incubation of red cells in APPD medium, the average IMP content increased to 5.30 +/- 2.33 mumol/g Hb, about 3.5 times that found in APP medium. At the same time ITP content was about 53.6% lower, that is 0.65 mumol/g Hb. In red cells incubated in APPD medium, penetration of 8-14C-adenosine decreased by 50%, and incorporation of this nucleotide into the pool of all free nucleotides also decreased by 18.2% as compared to red cells incubated in APP medium. It is concluded that IMP is probably formed directly from AMP gained by the phosphorylation of adenosine during its penetration.     

Comparison of renal damage by iodinated contrast or gadolinium in an acute renal failure rat model based on serum creatinine levels and apoptosis degree

This study was undertaken to compare renal damage, as determined by serum creatinine and degree of apoptosis, caused by iodinated contrast or gadolinium in an acute renal failure (ARF) rat model. Rats were divided into three groups; controls (n=3), a CT contrast medium group (n=9), and an MR contrast medium group (n=9). The CT and MR groups were further subdivided into three groups, namely, low, standard, and high dose subgroups. Renal function was evaluated by determining serum creatinine levels; before ARF, and 48 hr after ARF and contrast administration. Apoptosis was assayed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). No significant creatinine level differences were observed between the CT and MR groups (p=0.116). Degrees of apoptosis in the renal cortex and medulla were more severe in the CT contrast medium group than in the control or MR contrast medium group (p<0.05). The study shows that CT contrast medium did not aggravate renal function more so than MR contrast medium in this ARF rat model. However, apoptosis examination in the renal cortex and medulla indicated that CT contrast medium induced more severe apoptosis than MR contrast medium (p<0.05). We conclude that CT contrast medium can be used for renal imaging studies when subjects are well hydrated and preventive medication is administered.     

New medium for the production of cholera toxin by Vibrio cholerae O1 biotype El Tor.

A new medium that stimulates in vitro production of cholera toxin by Vibrio cholerae O1 El Tor (El Tor vibrios) was developed. The medium contains 0.5% NaCl, 0.3% NaHCO3, 0.4% yeast extract, and 1.5% Bacto-Peptone. El Tor vibrios were cultured in a stationary test tube at 37 degrees C for 20 h. The culture supernatant was assayed for cholera toxin by a reversed passive latex agglutination method. Most vibrios grown in this medium produced 10 to 20 times more toxin than in traditional syncase medium. The number of live vibrios at the end of culture was about 10(8)/ml in the new medium (AKI medium) and about 10(10)/ml in the syncase medium. As a result, each individual organism in the new medium should have produced as much as 1,000 times more toxin than in syncase medium. Sodium bicarbonate was found to be the most important factor in toxin production by El Tor vibrios in the new medium. We recommend this new medium because of its high yield of cholera toxin and its technical simplicity.