小鼠骨髓源性树突状细胞培养与冻存

背景:树突状细胞因其良好的抗原提呈功能及促T细胞增殖功能,成为肿瘤抗原的良好载体.获得大量的树突状细胞,对于肿瘤疫苗的研制具有重要作用.目的:对小鼠骨髓树突状细胞进行诱导、培养、扩增及冻存,并对比冻存后复苏细胞与未冻存细胞的细胞特性,寻找一种能够大量获得树突状细胞及有效储存的方法.方法:取小鼠骨髓细胞,在含重组小鼠粒-巨噬细胞集落刺激因子(10 μg/L)和重组小鼠白细胞介素4(5 μg/L)的完全培养基中对其进行诱导、培养及扩增.培养第6天,对培养获得的树突状细胞在加有冷冻保护剂DMSO的完全培养液中冻存.复苏后,在培养基中加入脂多糖、对细胞诱导,获得大量的成熟树突状细胞,最终获得的树突状细胞与未进行冻存的细胞在细胞活力、形态学、细胞表型、混合淋巴细胞反应等方面对比.结果与结论:复苏后,(82.2±4.73)%细胞存活,存活的细胞经脂多糖诱导后发育为成熟树突状细胞,在形态学、细胞表型及混合淋巴细胞反应等方面与未经冻存的树突状细胞差异无显著性意义.提示小鼠骨髓源性树突状细胞冻存复苏后,细胞生物学特性与未冻存的细胞无明显差别,用冻存的方法储存树突状细胞,能够避免在不同时期应用细胞时反复进行培养,可以获得大量的同质细胞.     

A simple method for the cryopreservation of human lymphocytes at −80C

A simple and inexpensive method for the freezing of human blood lymphocytes at −80 C is described. Lymphocytes were frozen in either polystyrene tubes or polyolefin bags. Optimal recovery of cells was obtained when 5% DMSO and 62% plasma were used as cryoprotectants. Viable cell recovery, as assessed by response to phyto hemagglutinin (PHA) and pokeweed mitogen (PWM), was greater than 80% after 60 days of storage. In addition, percentages of T and B lymphocytes were essentially unchanged. The excellent viable cell recovery in polyolefin bags suggests that it is possible to cryopreserve large numbers of lymphocytes in a single container.     

一种简单实用的单细胞分离技术

背景:基于单细胞水平的研究是干细胞、肿瘤干细胞研究的发展趋势.许多研究者常常因为无法轻易获得足够数量的单细胞而在研究时望而却步.掌握简单有效的单细胞分离技术为准确、定量的单细胞水平研究奠定基础.目的:介绍一种简单有效的单细胞分离技术.方法:在显微操作法的基础上改进,利用改制小号的静脉注射静脉注射针及微量移液器组合制成单细胞分离种植装置并采取双手操作法进行单细胞分离.采用染色细胞分离的方法对该技术分离单细胞的效率进行验证.结果与结论:单细胞分离的成功率达到96.5%.利用该技术分离单细胞具有取材容易、制作简单、操作直观可靠、分离成功率高、易于推广普及的特点.分离的单细胞可直接用于进一步的研究.     

一种改良的外周血干细胞深低温冷冻保存方法应用研究

目的　考察改良的外周血干细胞深低温冷冻保存方法。方法　采用两步离心法获得患者自体贫血小板血浆 ,在冷冻保护液中用自体贫血小板血浆代替药品人血清白蛋白。解冻后预防性地向袋中注入袋内体积 2 0 %的ACD A。结果　 17例患者 2 5次采集并深低温冷冻保存 ,解冻后活细胞回收率为 90 2 8%± 5 38% ;集落的回收率90 5 8%± 9 76 %。快速输入患者体内后无任何不良反应 ,全部较快地获得造血重建。结论　改良的外周血干细胞深低温冷冻保存方法能有效地保存干细胞 ,降低了冷冻经费 ,使输注更加安全。     

A simple two-step culture system for the large-scale generation of mature and functional dendritic cells from umbilical cord blood CD34+ cells

BACKGROUND: In vitro generated dendritic cells (DCs) are widely used as adjuvants in cancer immunotherapy. The major sources for DC generation are monocytes and CD34+ cells. CD34+-derived DCs are less frequently used in clinical applications because it requires complex generation methods. Here a simple method for the large-scale generation of mature functional DCs from umbilical cord blood–derived CD34+ cells is described.
STUDY DESIGN AND METHODS: CD34+ cells were first expanded with a combination of early acting growth factors in a medium containing autologous plasma. In the second step the DC precursors were further either enriched by plastic adherence or sorted on a cell sorter and differentiated as DCs. DCs generated by both methods were compared for their morphology, phenotype, and different functional variables.
RESULTS: This culture system provided a large-scale expansion of CD34+ cells giving a mean fold increase of 615. The majority of the expanded cells were interstitial DC precursors, that is, CD14+-positive cells. In vitro generated immature DCs could be matured into functional DCs by appropriate maturation stimuli. DCs generated by the plastic adherence method had a better cytokine profile and strong mixed leukocyte reaction compared to those generated by cell sorting.
CONCLUSION: A two-step culture system provides a large-scale expansion of CD34+ cells with a preferential lineage commitment toward CD14+ cells. Enrichment of these precursors with a simple plastic adherence technique results in generation of large numbers of mature, functional DCs. This method of in vitro DC generation will have applications in cancer immunotherapy.     

人外周血树突状细胞低温保存的优化研究

目的探寻冻存树突状细胞(DCs)优化的冷冻保护剂组合。方法配制含不同浓度二甲基亚砜(DM-SO)的3种冷冻保护剂组合,A组:5%DMSO+6%羟乙基淀粉(HES)+4%人血清白蛋白(HAS);B组:10%DMSO+40%FCS;C组:12%DMSO+40%FCS,比较3组冷冻保护剂对人外周血CD14+单个核细胞诱导产生的成熟树突状细胞(mDCs)的冻存效果:采用两步法将mDCs冻存于-80℃冰箱过夜后转移至-196℃液氮气相中放置24 h,再将冻存的mDCs复苏后继续培养,并检测、比较冻存前后DC的形态、存活率、细胞表型及其对同种异体T细胞刺激活性的差异。结果 3组不同组合冷冻保护剂冷冻保存的mDCs复苏后其存活的细胞的形态没有发生明显改变,仍保留其成熟表型,并具备对T细胞的刺激活性。结论 3种不同浓度的DMSO冷冻保存mDCs,5%DMSO+6%HES+4%HSA组合更适宜。     

A simple controlled-rate freezing method without a rate-controlled programmed freezer provides optimal conditions for both large-scale and small-scale cryopreservation of umbilical cord blood cells

BACKGROUND: Umbilical cord blood (CB) is being used as a source of alternative HPCs for transplantation with increasing frequency. The goal of CB banks for unrelated transplantation is to provide good quality-controlled CB units that can be transplanted for HPCs into the largest possible number of patients. STUDY DESIGN AND METHODS: Large CB samples in freezing bags wrapped with insulators and small samples in cryotubes placed into double styrene-foam boxes were cryopreserved at -85 degrees C without a rate-controlled freezing machine, followed by storage in the liquid phase of nitrogen. After thawing these cells, the viability and recovery of cells, as well as the recovery rate of HPCs such as CD34+ cells, CFU-GM, and total CFU were evaluated. RESULTS: Measurement of the freezing rate in CB bags and cryotubes demonstrated that this simple method for cryopreservation of CB cells provided optimal conditions for both large-scale and small-scale cryopreservation. Recovery of CB progenitor cells after cryopreservation was also shown to be potentially acceptable when evaluated with CD34+ cells, CFU-GM, and total CFU. These results were comparable to the method using a rate-controlled programmed freezer. CONCLUSIONS: A simple method for cryopreservation of CB cells without a rate-controlled programmed freezer could provide a sufficient-enough potential for the transplantability of HPCs after thawing.     

A large number of mature and functional dendritic cells can be efficiently generated from umbilical cord blood–derived mononuclear cells by a simple two‐step culture method

BACKGROUND: Advances in the past two decades in dendritic cell (DC) biology paved the way to exploit them as a promising tool in cancer immunotherapy. The prerequisite for DC vaccine preparations is large‐scale in vitro generations of homogeneous, mature, and functional DCs. Frequent improvements are being made in the existing in vitro DC production protocols to achieve this goal. In our previous study we reported a large‐scale generation of mature, functional DCs from umbilical cord blood (UCB) CD34+ cells. Here we report that this method can be used for the efficient generation of DCs from UCB mononuclear cells (MNCs) and thus the hematopoietic stem cell isolation step is not essential. STUDY DESIGN AND METHODS: MNCs or CD34+ cells isolated from the same cord blood (CB) samples were used for the generation of DCs. DCs were characterized for morphology, phenotype, and functional assays including antigen uptake, chemotaxis, and mixed leukocyte reaction. Similarly DCs generated from the MNCs of same fresh and frozen CB units were compared. RESULTS: The morphologic, phenotypic, and functional characterization of the DCs generated from various sets show that they were comparable in nature irrespective of the starting population used. CONCLUSION: We conclude that the CD34+ isolation step is not essential for the generation of mature, functional DCs and thus can be eliminated. More importantly, we show that DCs can be generated with equal efficiency from the MNCs of frozen CB units. Our culture method will be useful for exploiting the potential of UCB as an additional source for allogeneic DCs in the clinical settings.     

A simple method for high-quality frozen red cells in blood group serology

A high-glycerol method for long-term storage of red cells for use in blood group serology testing is described. Total freezing-thawing-washing hemolysis was 8.05 +/- 0.3 percent. Enzyme treatment could be performed before freezing or after reconstitution. The cells performed well with manual and automated techniques. No changes in agglutinability were noted during 10 days of liquid storage at 4 degrees C following thawing.     

A simple method for the cultivation of rat liver cells.

A simple method to culture rat liver parenchymal cells is described. Minced liver tissues were dissociated with no pre-perfusion by a newly developed enzyme, bacterial neutral proteinase, and cultured in the medium consisting of 10% fetal calf serum and 90% of a new synthetic medium, DM-153. By the adequate technique of primary dissociation and first subcultivation, parenchymal cells were selected. Cultivation in arginine-free medium was also useful in selecting them. The cultured cells exhibited activities of certain enzymes similar to those of liver cells in vivo. Cell strains have been successively established from liver tissues of embryo, suckling and adult rats. Liver cells, however, cultured in this way, can also be used for experiments in the early stage of serial cultivation.     

A simple method to estimate functional residual capacity in mechanically ventilated patients

Objective The aim of the present study was to evaluate a simplified method for FRC measurement.Design: Accuracy and precision of the method were assessed in a physical lung model; reproducibility was tested in 10 mechanically ventilated patients. In each patient FRC was measured at three PEEP levels.Setting: Post-operative intensive care unit in a university hospital.Measurements and results Gas flow, CO₂ concentration, and O₂ concentration were measured during in- and expiration by pneumotachography, a mainstream capnometer and a sidertream O₂-analyser. For FRC-measurement inspiratory O₂ concentration was changed by 30%. FRC was determined as mean value of a N₂ washout and N₂ washin procedure. Evaluation of this method in a lung model shows a good correlation between FRC set in the lung model and FRC measured (FRC measured =1.028*FRC model+22.92 ml;r ²=0.957;n=30). The mean difference was 4.4% of FRC-reference (range –8.4% to +21.7%). Duplicate determinations in 10 mechanically ventilated patients differed by an average of –2.7% (range –30.1% to +27.3%).Conclusion Our results suggest that the proposed method can be used in daily clinical work.     

Strategies used in post-operative pain assessment and their clinical accuracy

Our knowledge about the content of strategies used by staff members in a surgical recovery unit for assessment of post-operative pain is fairly limited. The aim of the present study was to describe variations in the content of strategies used by nurses and physicians in practical clinical pain assessments and to evaluate the clinical accuracy of the strategies used. Critical care nurses (n = 30), physicians (n = 30) and postsurgical patients (n = 180) comprise the respondents. Applying a phenomenographical approach, interview data were tape-recorded during 180 clinical pain assessments. The pain assessments were related to comparative bedside pain ratings (Visual analogue Scale, VAS), both by staff members and post-operative patients. The recorded interviews were analysed to describe variations in ways of assessing pain. Pain assessment strategies were established by combining categories describing the impact of experience and categories of assessment criteria. The present observations, if included in the education of clinical staff members, could increase the understanding and thereby the quality of the pain assessment process.     

A method for the cryopreservation of red blood cells using hydroxyethyl starch as a cryoprotectant

A simple, quick, inexpensive and long term method of cryopreserving human red cells using hydroxyethyl starch (HES) as a cryoprotectant is described. No sophisticated equipment or skilled labour is required. As HES is a plasma expander, it does not have to be removed prior to transfusion, thereby obviating the requirement for a washing stage. This allows 60 units/h to be prepared. As the microbiological integrity of the system is maintained, there is potential for prolonged storage post-thaw at 4 degrees C. Recoveries of 99% and 30 min saline stabilities in excess of 91% have been consistently achieved. P50O2 and 2,3DPG values are not significantly decreased.     

A simple slide-rule method for the assessment of renal tubular reabsorption of phosphate in man

The activity of renal tubular reabsorption of phosphate in man is best expressed as the ratio of the maximum rate of reabsorption to the glomerular filtration rate (TmP/GFR). A slide-rule method based on existing data is described for the derivation of TmP/GFR from values of phosphate and creatinine concentrations in single samples of plasma and urine. This is a simple method which is suitable both for research and for clinical purposes.     

Phenotypical and functional characterization of clinical grade dendritic cells

Dendritic cells (DC) are the professional antigen presenting cells of the immune system. Therefore, several clinical studies have been initiated in which tumor antigen-loaded DC are used as a vaccine to boost an immune response against malignant tumors in patients with cancer. A prerequisite for DC used in these vaccination studies is not only that they are grown under "Good Manufacturing Practice" but equally important that they retain their functional properties. In an extensive study, various conditions were tested to optimize the maturation and yield of DC grown for clinical use. DC grown in XVIVO-15 medium supplemented with 5% HS yielded the best results, morphologically and phenotypically. Mature DC expressed significant amounts of mature DC markers (CD83) and the costimulatory molecules CD80 and CD86. It was shown that mature and immature DC can be frozen and retain their phenotype and function after thawing. These clinical grade DC secreted high levels of the chemokines dendritic cell chemokine 1 (DC-CK1), interleukin-8 (IL-8), macrophage-derived chemokine (MDC), and thymus and activation-regulated chemokine (TARC). This implicates that these DC can attract na?ve T and B cells as well as natural killer cells and memory T cells. Finally, to test their migratory capacity in vivo, (111)In-labeled DC were injected into tumor-free lymph nodes of patients with melanoma. Autoradiographic analysis of the dissected lymph nodes indicated that these DC could migrate into the T cell area of adjacent lymph nodes. In conclusion, a culture procedure was established to generate large numbers of monocyte-derived immature and mature DC that retain their morphologic, phenotypic, and functional characteristics in vitro and can be visualized in situ.     

A simple method for estimating compliance

In intensive care medicine, pulmonary compliance is one of the very helpful diagnostic indices. Because of technical difficulties, however, the measurement of pulmonary compliance is often reduced to a rough guess of the compliance of the total respiratory system. The technical problems can be overcome using a computer to solve the basic equations with the least-squares fit (LSF) method. Unfortunately, this method requires such a long calculation time that bedside breath-by-breath calculations are impracticable on small computers. A simple computer algorithm (mean-values method) was therefore developed and compared to the LSF method. Compliance values calculated by either procedure were practically identical in ventilated patients. However, by reducing computing time to 30% of the LSF method, our mean-values algorithm enabled real-time estimation of compliance breath-by-breath.