The preservability of rosette-forming blood cells in various culture media

Rosette-forming cells of the peripheral human blood are preservable in the cell culture media McCoy's 5a, RPMI 1640 and supplemented Eagle-MEM (59) at 20 degrees C for 3 days, if 1 part blood is mixed with 2 parts of one of the culture media.     

The production of differentiation autoinducing activity by WEHI-3B D+ leukemia cells

We studied the differentiation autoinducing activity in WEHI-3B D+ cell-conditioned medium (WCM). After culturing 10(6)/ml WEHI-3B D+ cells in RPMI-1640 medium without fetal calf serum (FCS) for 4 days, the supernatant was collected. The medium, concentrated 50-fold by YM-5 membrane filtration, was fractionated by gel exclusion on Ultrogel AcA44. We evaluated the effect of each of the four fractions on differentiation in WEHI-3B D+ cells by morphological, functional, and cytochemical criteria after adding the fractions to liquid or soft-agar cultures of 10(3) cells in 1 ml RPMI-1640 medium containing 10% FCS; the experimental cultures contained 10% of the fractions, with a control for each without the fraction. The growth of WEHI-3B D- cells in culture was inhibited by the addition of fraction P only (mol. wt. 10,000-20,000 daltons). In these same cultures, the cells were granulocyte-like, strongly positive for naphthol ASD chloroacetate esterase, and had phagocytic activity. Colonies grown in agar culture with fraction P also exhibited a peripheral halo of loosely dispersed cells around a central aggregate. Fraction P contained neither granulocyte colony-stimulating activity nor burst-promoting activity. These results suggest that fraction P contains differentiation autoinducing factor that is different from granulocyte colony-stimulating factor or interleukin 3.     

In vitro cultivation of infective larvae of Dirofilaria immitis

Infective larvae (L3) of Dirofilaria immitis were cultivated in various synthetic media. L3 obtained from Aedes aegypti mosquitoes by mass dissection were inoculated into the media, viz: RPMI 1640 medium with 10 per cent human AB serum (medium-A); Tc-199 medium with organic acids, sugars of Grace's insect medium and 10 per cent foetal calf serum (FCS) (medium-B); and Grace's insect medium with 10 per cent FCS (medium-C). Maximum survival of 26 days was found in medium 'A'. Approximately 80 per cent healthy larvae moulted after 72 hours. They increased in size from an initial average length of 1050 microns and 25 microns width to an average 1600 microns length and 35 microns width. Blunting and rounding of anterior ends were conspicuous.     

Preparation and culture of dispersed avian pituitary cells, and age-related changes in donor pituitary weight and growth hormone content

Techniques were perfected for the enzymatic dissociation of chicken pituitary glands and a number of factors evaluated for their effects upon growth hormone (GH) production by dispersed chicken pituitary cells in culture. Age-related changes in donor pituitary weight and GH content were also determined. A procedure involving digestion of minced glands with a solution of 0.1% trypsin in S-MEM tissue culture medium (0.1% BSA) for 1 hr at 37 degrees under an atmosphere of 5% CO2-95% air yielded greater than 2.0 X 10(6) cells per gland with 80-90% viability. Five tissue culture media (D-MEM, alpha-MEM, RPMI 1640, Med-199, Earle's salts), two serum sources (calf serum (CS), horse serum (HS), and two levels of serum (5, 20%) were tested for their ability to support GH synthesis over 4 days in culture. Additionally, two culture regimes (continuous culture vs daily media changes) were evaluated for their effects on GH production. alpha-MEM resulted in the numerically highest net GH synthesis (over starting cell content), although not statistically different from RPMI 1640 or Earle's salts. Neither serum type nor percentage was significant; therefore the lower serum percentage (5) was adopted for future studies. Culture regime significantly altered the proportion of secreted vs stored hormone harvested at the end of the culture period. Changing media daily resulted in a 40% reduction in final cell GH content compared to continuous culture, whereas total cumulative media GH was approximately 39% greater (P less than 0.01). Pituitary weight increased with age until approximately 9 weeks, whereas GH content plateaued earlier, at 5 weeks of age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular epidemiology of malaria in Cameroon. XXIII. Experimental studies on serum substitutes and alternative culture media for in vitro drug sensitivity assays using clinical isolates of Plasmodium falciparum

Correlation studies on the in vitro drug response of field isolates of Plasmodium falciparum and molecular markers for drug resistance are becoming important as many malaria control programs abandon monotherapies and resort to combination therapies. The standardization and optimization of the in vitro drug sensitivity assay are one of the prerequisites for validating molecular markers in the field. The present study was designed to assess and compare the growth of freshly obtained isolates for at least the first erythrocytic cycle in various culture media and determine the in vitro response to chloroquine in alternative media. Parasite growth was consistently higher in Dulbecco's modified Eagle's medium (DME)-human serum, Iscove's modified Dulbecco's medium (IMDM)-human serum, RPMI 1640 medium-goat serum, and a serum-free medium containing 1:1 (v/v) mixture of IMDM and F-12 supplemented with an ammonium sulfate fraction of adult bovine serum than in RPMI 1640 medium-human serum mixture. The level of chloroquine response determined in human serum-supplemented DME, IMDM, and RPMI 1640 media did not differ significantly (P > 0.05) from the control (RPMI 1640-human serum). This study suggests that alternative media may be used to optimize parasite growth during the critical initial phase of transition from in vivo to in vitro conditions. The capacity of these media to support long-term cultivation of P. falciparum requires further investigation.     

Effect of different media on long-term cultivation of human synovial macrophages]

Cells of rheumatoid synovial tissue were bred in dispersion culture. In order to investigate the influence of three different culture media on the maturation and vitality of synovio-macrophages, Ham F10 (+fetal calf serum) and RPMI 1640 (+pooled AB Rh+ human serum) were compared with the serum- and cytokin-free solution AIM V. Combining light microscopic, transmission electron, and scanning electron microscopic methods (backscatter-mode), the enzyme and antibody features of synoviocytes were detected. The ratio of macrophages were determined in the light microscope by marking with CD 14. The functional maturation was shown by the amboceptor test. This study demonstrates that RPMI 1640 (+AB Rh+ human serum) is favorable for cultivating human synoviomacrophages. Under serum-free conditions AIM V is especially appropriate.     

Use of urine samples from healthy humans, nephritis patients or other animals as an alternative to foetal calf serum in the culture of Leishmania (L.) donovani in vitro

The effect of supplementing in-vitro cultures of Leishmania donovani with urine was investigated. The parasites were isolated from Bangladeshi patients with visceral leishmaniasis. The urine samples used were collected from healthy human donors, patients with nephrotic syndrome, diabetic nephritis (DN) or diabetes mellitus, a dog and a cow. Promastigotes from blood-agar cultures were inoculated into RPMI-1640 basal medium with 10% heat-inactivated foetal calf serum (FCS) and/or 1%-20% urine. The parasites were then counted in a haemocytometer, on days 2, 4, 5, 6, 7, 8, 10, 12 and 14 post-inoculation. From day 4, the numbers of parasites/ml in cultures containing 5% healthy-human urine but no FCS were at least as high as those in cultures containing 10% FCS but no urine (P = 0.191). The wet weights of parasites harvested from mass cultures of the parasites in RPMI-1640 plus 5% healthy-human urine and in RPMI-1640 plus 10% FCS were practically the same. Multiplication of the parasites in the presence of 5% urine from a DN patient was significantly greater (P < 0.01) than that seen with other urine samples at the same concentration or with 10% FCS. The multiplication seen with 8% canine urine was almost the same as with 5% healthy-human urine. Parasites could be maintained in RPMI-1640 plus 5% healthy-human urine for at least 40 days, sub-culturing every 4 days. Urine may be a better and much cheaper stimulant of Leishmania multiplication in vitro than FCS.     

Microculture for the isolation of Leishmania parasites from cutaneous lesions -- Sri Lankan experience

Novy, McNeal and Nicolle (NNN) medium and Evans' modified Tobie's medium are two conventional media for the isolation of Leishmania parasites in in-vitro cultures. Both are biphasic, with a solid layer of blood agar, and are normally prepared in glass test-tubes. In Sri Lanka at least, a monophasic microcapillary culture, based solely on RPMI 1640 medium supplemented with foetal calf serum, has been found simpler, more economical and more sensitive, for the isolation of L. donovani from skin lesions, than the use of Evans' modified Tobie's medium.     

Development of onchocerca lienalis and O. volvulus from the third to fourth larval stage in vitro

Third-stage larvae of Onchocerca volvulus and O. lienalis were observed to molt to the fourth stage in various cell-free in vitro systems. The percentage of O. lienalis completing the molt was similar in the three culture media and two gas phases tested ranging from 44.8% (1:1 IMDM:NCTC + 5% CO2: 95% N2) to 56.7% (L-15 + 5% CO2: 95% air). Percent molting in O. volvulus ranged from 0% (F12(K) + 5% CO2: 95% N2) to 33.3% (L-15 + 5% CO2: 95% N2). All media were supplemented with either 20% FCS or 20% horse serum. Molting by O. lienalis occurred on days 2-5 in culture. Molting by O. volvulus was observed as early as day 5 and as late as day 10. Incomplete casting of the third-stage cuticle was frequently observed in O. volvulus. Larvae of both species entered a lethargus 24-48 hours prior to the onset of molting. Maximum survival in culture was 42 days for O. lienalis and 25 days for O. volvulus. Significant growth of larval O. lienalis was noted early in the culture period, but neither species continued development to the fifth stage.     

不同血清浓度及介质对体外培养小鼠成纤维细胞L929细胞系生长曲线和细胞形态的影响

目的：通过探讨不同血清浓度及介质对体外培养小鼠成纤维细胞L929细胞系生长曲线和细胞形态的影响，了解它们对成纤维细胞体外生存和生长的情况。方法：采用体外细胞培养技术进行小鼠成纤维细胞L929细胞，并采用磺基罗丹明B（sulforhodamineB，SRB）酶标仪法（A490nm波长下）测量各孔的吸光值（A值）并转换为小鼠成纤维细胞L929细胞系生长曲线；同时，分别采用HE（苏木精-伊红）染色法、、吖啶橙荧光染色和Hoechst33342荧光染色法进行小鼠成纤维细胞L929细胞系形态学观察。结果：小鼠成纤维细胞L929细胞系不同浓度的血清分别在3种不同的介质（RPMI1640培养基、0．9％生理盐水和5％葡萄糖生理盐水）中表现出不同的生长和生存情况及细胞形态学改变。其中，在不同血清浓度（0％、10％、20％、30％、40％、50％、60％、70％、80％、90％、100％）的同一介质中，血清浓度为0％和100％浓度组的小鼠成纤维细胞L929细胞生长明显抑制及吸光度（A）值呈逐步减少的趋势，而除此之外的其它血清浓度组对小鼠成纤维细胞L929细胞的影响，基本表现为随着血清浓度的升高，吸光度（A）值呈逐步增加及促进细胞生长的趋势。而某些相同血清浓度在不同介质中对小鼠成纤维细胞L929细胞系生长和生存及形态学的有利影响，则基本表现为RP—M11640培养基作为介质的细胞培养效果优于0．9％生理盐水，而0．9％生理盐水作为介质的细胞培养效果又优于5％葡萄糖生理盐水。结论：不同血清浓度及介质对体外培养小鼠成纤维细胞L929细胞系生长和生存及细胞学形态均有明显影响。提示在进行体外细胞培养时应充分考虑细胞培养介质及所添加血清的浓度。     

抗包虫药物的实验研究——Ⅰ.吡喹酮体外抗细粒棘球蚴原头节的作用

在20％小牛血清-RPMI1640中,吡喹酮对体外培养的细粒棘球蚴原头节有较强的杀灭作用,主要表现为外翻、皮层肿胀、泡状物形成、破裂,以及头钩排列紊乱和脱落等,24h后即出现死亡,3天内的死亡率达90％以上。吡喹酮对培养在囊液中的原头节亦有杀灭作用,但对培养在RPMI1640和HBSS中的则无。进一步观察的结果表明,吡喹酮在体外抗原头节的作用需赖于有蛋白质的存在。此外,小鼠口服吡喹酮后,其血清有抗原头节作用,主要成份为吡喹酮原药。     

Storage of unprocessed G-CSF-mobilized whole blood in a modified Leibovitz's L15 medium preserves clonogenic capacity for at least 7 days

Autologous stem cell transplantation using unprocessed, G-CSF-mobilized whole blood (WB) is a simple, cost-reducing procedure and supports high-dose chemotherapy regimens not exceeding 72 h. Thereafter, clonogenic capacity rapidly decreases if routine anticoagulants are used for storage. In order to increase clinical applicability, we investigated the requirements for optimal preservation of unprocessed WB for 7 days. During storage at 22 degrees C in CPDA-1, a decrease in pH was noted, which was at least partially responsible for the low recovery of clonogenic cells. Subsequently, WB cells were stored in various cell culture media (RPMI 1640, alpha-MEM, X-VIVO15, CellGro SCGM and Leibovitz's L15 medium) containing either serum, serum-free substitutes or no additives. Leibovitz's L15 showed significantly better CFU-GM recoveries than the other media. Using a calcium-free modification of L15 medium (added 3:10 to WB), 94 +/- 24% of CD34(+) cells, 41 +/- 14% of BFU-E, 56 +/- 17% CFU-GM and 90 +/- 14% of LTC-IC were preserved during storage for 7 days at 22 degrees C. Storage at 4 degrees C was also feasible, but showed less optimal recoveries of 52 +/- 29% (CD34), 32 +/- 10% (BFU-E), 13 +/- 7% (CFU-GM) and 58 +/- 9% (LTC-IC). The expression of CD38, Thy-1, c-kit, AC133, L-selectin and CXCR4 on CD34-positive cells remained unchanged. In conclusion, a modified Leibovitz's L15 medium better meets the metabolic requirements of a high-density cell culture and allows safe storage of G-CSF mobilized WB for at least 7 days. The results encourage further exploration of WB transplants stored for 7 days for clinical use.     

Islet amyloid polypeptide promotes beta-cell proliferation in neonatal rat pancreatic islets

Aims/hypothesis: We aimed to clarify the role of islet amyloid polypeptide, which is expressed at early embryonic onset, in the proliferation and cell death of neonatal islet cells. Methods: Fetal islets were prepared from pregnant rats on gestational day 21. Islets were cultured in RPMI 1640 (11.1 mmol/l glucose) + 10 % fetal calf serum (FCS) for 48 h, followed by a 24-h culture period in RPMI 1640 (5.6 mmol/l glucose) + 1 % FCS. The islets were then exposed to rat islet amyloid polypeptide (1–10 nmol/l) for 24 h. Results: Iselt amyloid polypeptide increased islet DNA synthesis (dpm/μg of DNA · 6 h) (control 1 % FCS: 3634 ± 662; 1 nmol/l 6347 ± 1535; 10 nmol/l 5157 ± 769; p < 0.05 islet amyloid polypeptide vs control). In accordance with this, a doubling of the autoradiographic labelling index was seen in immunocytochemically stained islet beta cells after exposure to 1 and 10 nmol/l islet amyloid polypeptide. Islet amyloid polypeptide at 1 nmol/l increased the islet insulin content (202 ± 25 % of control; p < 0.01) and the 24-h medium insulin concentration (1 nmol/l islet amyloid polypeptide: 143 ± 19 % of control; p < 0.05) but at 10 nmol/l islet amyloid polypeptide these changes did not attain statistical difference. Islet amyloid polypeptide did not have any marked effect on the islet cell death frequency, suggesting that islet amyloid polypeptide is a more potent promoter of proliferation than of programmed cell death. Conclusion/interpretation: Our data indicate islet amyloid polypeptide is a potential regulator of proliferation in neonatal pancreatic islet cells, an effect which can partly be attributed to the proliferation of beta cells. [Diabetologia (2001) 44: 1015–1018] Received: 14 March 2001 and in revised form: 7 May 2001     

Serum deprivation enhances DNA synthesis of human hepatoma SMMC7721 cells

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肾小球系膜细胞白细胞介素-6基因表达与白细胞介素-13的关系

目的探讨白细胞介素-13(IL-13)对于体外培养大鼠系膜细胞白细胞介素-6(IL-6)分泌及其基因表达的影响,以研究IL-13对肾小球疾病状态下肾脏系膜细胞炎症反应的抑制作用。方法用酶联免疫吸附法(ELISA)测定系膜细胞IL-6分泌量,用逆转录聚合酶链反应(RT-PCR)检测系膜细胞IL-6mRNA表达。结果正常培养条件下系膜细胞IL-6mRNA表达及IL-6分泌水平较低,脂多糖(LPS)可刺激系膜细胞IL-6mRNA的表达及IL-6分泌水平,而IL-13则呈剂量依赖性地抑制LPS诱导的系膜细胞IL-6分泌及其mRNA表达。结论IL-13抑制LPS诱导的体外培养的系膜细胞IL-6产生,故可能对于肾小球疾病状态下肾脏的系膜细胞炎症反应具有抑制作用。     

X线照射泡球蚴原头节的体外实验研究

目的探讨X线对体外培养的泡球蚴原头节的杀伤作用。方法无菌采集子午沙鼠体内的泡球蚴中含原头节的囊液,将其加入RPMI 1640培养液中培养。原头节体外培养3 d后分装至培养瓶中,每组10瓶,每瓶约含10 000个原头节,设空白对照组、低剂量组(15 Gy和30 Gy)、中剂量组(45 Gy和60 Gy)、高剂量组(75 Gy和90 Gy)、阿苯达唑组(2 500 ng/ml)、45 Gy X线+2 500 ng/ml阿苯达唑组和75 Gy X线+2 500 ng/ml阿苯达唑组。X线照射剂量率为200 cGy/min,源皮距为100 cm。体外培养第4天开始照射,每组共照射3次,每次间隔1 d。首次照射后第1天开始每天取原头节培养液,0.1%伊红染色,光镜下计数每100个原头节中着色原头节数目,每组计算300个原头节的平均死亡率,直至实验组原头节全部死亡为止。同时光镜下观察经X线照射后原头节的变化。结果不同放射剂量组的原头节死亡率与空白对照组间的差异均有统计学意义(P<0.05)。阿苯达唑组原头节死亡率与放射线联合阿苯达唑组间的差异均有统计学意义(P<0.05),且显著高于空白对照组(P<0.05)。其中,X线...     

Improvement of growth of Plasmodium falciparum fresh clinical isolates by using an established serum-free medium, GIT

In the present study, we have tried to establish continuous cultures of fresh clinical isolates of P. falciparum by using a serum-free medium, GIT. To examine the ability of GIT to support the parasite growth, the growth of various P. falciparum isolates including two laboratory strains of P. falciparum, FCR3 and K1 was compared in both of GIT and RPMI 1640 medium supplemented by 10% human serum (RPMI-HS). Growth rates of various P. falciparum expressed as fold increases were compared in GIT and RPMI-HS, and the maximum growth rates of P. falciparum were 72 in GIT and 35 in RPMI-HS during the culture for 8 days. Growth rate of the clinical isolates varied individually in both culture media, with average growth rates of parasites being 15.9 in GIT and 8.8 in RPMI-HS, respectively (not significant). Growth rates of FCR3 and K1 strains were 28.0 and 6.6 in GIT, and 10 and 7.5 in RPMI-HS. After 30 days culture of P. falciparum in GIT, 9 of 12 clinical isolates still continuously propagated but other three isolates disappeared. Despite variation of the P. falciparum isolates in their abilities to multiply in GIT, our experiments suggested that GIT is useful for culture of fresh clinical isolates of P. falciparum that are derived from geographically distinct areas as well as laboratory strains used commonly in laboratory research.     

血清饥饿法用于细胞周期同步化的方法学研究

目的探讨血清饥饿法进行细胞周期同步化实验的影响因素。方法用无血清和低血清浓度培养基饥饿Anip973和AGZY83-a两种人肺腺癌细胞系，分别在培养48h、72h和5d时收集各组细胞，用流式细胞分析仪分析细胞周期各时相细胞百分数。结果Anip973细胞系在含O．2％FBS的RPMI1640培养基中培养5d得到理想结果，G0-G1期细胞百分比高达84．19％。AGZY83-a在含O．2％FBS的RPMI1640培养基中培养48h也获得较满意的结果，G0-G1期细胞百分比达61．30％。结论细胞类型、血清浓度和饥饿时间是血清饥饿法进行细胞周期同步化实验成功与否的影响因素。可应用血清饥饿法使不同细胞系同步于G0-G1期。     

Liquid preservation of human neutrophils stored in synthetic media at 22 degrees C: controlled observations on storage variables

The purpose of this study was to use a chemically defined medium to identify essential substances and optimal conditions for the liquid storage of neutrophils at 22 degrees C. Several commercially available synthetic media were evaluated: L-15, McCoy's 5a, M199, minimum essential medium, Dulbecco's MEM, NCTC135, and RPMI 1640. Proteins, glucose, pH, and neutrophil concentration were systematically studied. Neutrophils were harvested by centrifugal cell separators or phlebotomy, and their maintenance was evaluated by monitoring cell counts, dye exclusion, phagocytosis, bacterial killing, and chemotaxis. Neutrophils stored equally well in all synthetic media except L-15; however, chemotaxis was poorly maintained in synthetic media as compared with autologous plasma. RPMI 1640 was arbitrarily selected as a basal medium to evaluate storage variables. RPMI 1640 supplemented with albumin to a concentration of 1% improved chemotaxis and was equivalent to plasma as a storage medium with regard to the in vitro functions tested. Cohn fractions IV-1, IV-4, and gamma globulin were not effective substitutes for albumin. Glucose is essential for neutrophil storage; its absence from the medium correlated with poor cell function. Optimal glucose requirements depend on the cell concentration. High glucose concentrations were toxic to neutrophils; at 1,000 mg/dL, chemotaxis was depressed by 58%. Glucose utilization was dependent on the initial pH of the medium and on the cell concentration. A wide range of hydrogen ion concentrations was tolerated, and the optimum pH range was 7.2 to 7.8. Cell concentration is an important variable because it affects the pH of the medium as well as glucose utilization.     

Establishment,growth properties,and morphological characteristics of permanent human small cell lung cancer cell lines

Summary Cell lines from SCLC were established with a success rate of 43% from different metastatic sites of treated and untreated patients. All 6 SCLC cell lines grew as floating cell aggregates without substrate adherence. The degree of aggregation ranged from very tight spheroids to very loose sheets and chains. This gross morphological property showed a striking correlation to the PDT, with short PDTs in loose growing cell lines and long PDTs in tight growing cell lines. Cell size and nuclear features, i.e., chromatin pattern and nucleolar prominence, also seemed to correlate with the PDT and gross morphology. All SCLC cell lines had dense core granules by electron microscopical examination. Several different serum-free and serum-supplemented growth media were tested for their feasibility in estabilishing and permanently growing SCLC. Serum-free SIT medium and SIT 2.5 medium provided the best results in liquid culture. For semisolid SCLC cultivation, R10 medium was suprior to all other media tested. These cell lines are currently under intensive biochemical, molecular biological, and cytogenetical investigation in different laboratories and thus provide a tool for studying the biology of lung cancer.Abbreviations SCLC small cell lung cancer - FBS fetal bovine serum - R 10 Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS - D 10 Dulbecco's modified Eagle's medium supplemented with 10% FBS - SIT RPMI 1640 medium supplemented with selenium, insulin, transferrin - HITES RPMI 1640 medium supplemented with hydrocortisone, insulin, transferrin, estradiol, selenium - SIT 2.5 SIT medium supplemented with 2.5% FBS - PBS phosphate buffered saline - PDT population doubling time This work was supported by the SFB 215 of the German Research Society. The authors Thank Dr. Adi F. Gazdar for reviewing the morphology