Cryopreservation of lipoaspirates: in vitro measurement of the viability of adipose‐derived stem cell and lipid peroxidation

As the storage time of the fat tissue passes by, lipid peroxidation and creation of by‐products may take place. The objective of this study was to evaluate the cell viability and functional changes of adipose‐derived stem cells (ADSCs) in the cryopreserved lipoaspirates at different temperatures in accordance with lipid peroxidation. Lipoaspirates acquired from liposuction were divided into four different temperature groups and stored at 4°C, −20°C, −80°C, and −196°C. After isolating ADSC from each sample, gross cell morphology and cell viability were compared with doubling time and colony‐forming unit (CFU) formation ability. Acid value, that is, thiobarbituric acid value was measured to assess lipid peroxidation. No viable ADSC was observed in −20°C and −196°C samples for past 1 week and a superior number of the live cells were detected in the 4°C group compared with the −80°C group. However, the persistence of cell division and CFU formation after 1 week was only observed in adipocytes stored at −80°C. Lipid peroxidation mainly occurred at 4°C and −20°C storage samples. If the lipoaspirates were planned to be cryopreserved, it is advised to store at −80°C. However, the number of actually functional ADSCs is very low. Furthermore, even in the cryopreserved status, continuous lipid peroxidation and by‐product creation took place, suggesting shorter preservation period as possible in the clinics.     

Influence of mature adipocytes on osteoblast proliferation in human primary cocultures

It has been shown that the bone loss occurring with aging in spongy bone is associated with a reduced osteoblastic bone formation and an increased volume of marrow adipose tissue. This observation suggests a relationship between cells from the osteoblastic and adipogenic lineages. The purpose of the present study was to evaluate the influence of mature adipocytes on osteoblastic proliferation and activity in a model of coculture. Human primary osteoblastic (hBOB) cells were derived from femoral bone explants collected in patients undergoing orthopedic surgery. Human stromal osteoblastic (hMSOB) cells were obtained from bone marrow samples collected by aspiration during orthopedic surgery. Extramedullary and medullary mature adipocytes (hAd) showing similar functions, except for their response to insulin, hAd were isolated from mammary adipose tissue collected in women undergoing tumorectomy. Cells were cocultured, with hAd being separated from osteoblastic cells (hBOB or hMSOB) by a porous membrane (0.4 microm). When hBOB cells were seeded on the upper side of the insert and hAd were floating on the lower side, cell contacts between the two cell types were possible through the pores of the membrane. At the end of the experiment, proliferation of the osteoblastic cells was evaluated by [(3)H]-thymidine incorporation and alkaline phosphatase (AP) activity was measured. After 20 h of coculture, proliferation of the hBOB cells was significantly decreased when compared with control hBOB (-40 +/- 6%, p < 0.05). To establish whether or not the influence of hAd on hBOB proliferation required intercellular communications, hAd and hBOB cells were cocultured far from the porous membrane. Six other independent experiments confirmed an inhibition of hBOB proliferation under both experimental conditions (p < 0.05): -35 +/- 7% with possible intercellular contacts, and -30 +/- 7% without any contact. In contrast, the proliferation of hMSOB cells was not significantly modified after coculture with hAd. In addition, the presence of hAd did not significantly modify the AP activity of hBOB (0.163 +/- 0.143 and 0.181 +/- 0.114 nmol/min per microgram of protein in controls and after coculture, respectively). No reproducible effect of hAd-conditioned medium was noted on hBOB- and hMSOB-cell proliferation or hBOB-cell activity. In conclusion, mature adipocytes induced an inhibition of hBOB-cells proliferation, probably mediated by a factor secreted by hAd. This effect may contribute to the age-related reduction of bone formation and bone loss.     

Interconversion potential of cloned human marrow adipocytes in vitro

Information on the interconversion potential of adipocytes and other end cells characteristic of the stromal fibroblastic cell lineages, key in the understanding of bone turnover in metabolic diseases such as osteoporosis, is limited. The object of the present study was: i) to isolate relatively pure populations of adipocytes from human bone marrow; ii) to clone single adipocytes from these populations; and iii) to examine in vitro the interconversion potential of the progeny of these single-cloned adipocytes between the osteogenic and adipogenic phenotypes. Adipogenic colonies were isolated from the low-density floating fraction of normal bone marrow cells cultured in adipogenic media for 4 days. Single adipocytes were isolated and cloned by limiting dilution. Cloned adipocytes were found to dedifferentiate into fibroblast-like cells, and subsequently to differentiate into two morphologically distinct cell types: osteoblasts and adipocytes in appropriate culture systems. The adipocytic phenotype was confirmed by morphology, oil red O staining, and immunocytochemistry using antiserum to aP2. The osteogenic phenotype was confirmed by alkaline phosphatase, osteocalcin immunostaining using specific osteocalcin antiserum, and formation of mineralized cell aggregates. These findings demonstrate the extent of plasticity between the differentiation of adipocytic and osteogenic cells in human bone marrow stromal cell cultures. We have shown the ability of isolated clonal adipogenic cells to redifferentiate into cells of the osteogenic and adipogenic lineage and the interconversion potential of human marrow stromal cells in vitro. These results provide further evidence that the osteogenic and adipogenic cells share a common multipotential precursor.     

From blood monocytes to adipose tissue-resident macrophages: induction of diapedesis by human mature adipocytes

Obesity has been suggested to be a low-grade systemic inflammatory state, therefore we studied the interaction between human adipocytes and monocytes via adipose tissue (AT)-derived capillary endothelium. Cells composing the stroma-vascular fraction (SVF) of human ATs were characterized by fluorescence-activated cell sorter (FACS) analysis and two cell subsets (resident macrophages and endothelial cells [ECs]) were isolated using antibody-coupled microbeads. Media conditioned by mature adipocytes maintained in fibrin gels were applied to AT-derived ECs. Thereafter, the expression of endothelial adhesion molecules was analyzed as well as the adhesion and transmigration of human monocytes. FACS analysis showed that 11% of the SVF is composed of CD14(+)/CD31(+) cells, characterized as resident macrophages. A positive correlation was found between the BMI and the percentage of resident macrophages, suggesting that fat tissue growth is associated with a recruitment of blood monocytes. Incubation of AT-derived ECs with adipocyte-conditioned medium resulted in the upregulation of EC adhesion molecules and the increased chemotaxis of blood monocytes, an effect mimicked by recombinant human leptin. These results indicate that adipokines, such as leptin, activate ECs, leading to an enhanced diapedesis of blood monocytes, and suggesting that fat mass growth might be linked to inflammatory processes.     

压应力对脂肪细胞活性的影响

目的 探讨压应力对脂肪细胞活性的影响,为提高自体脂肪移植的成活率及自体脂肪移植的临床应用提供参考.方法 将同等条件获取的脂肪颗粒随机分为5组,分别为对照组(0 kPa)、25 kPa组、50 kPa组、75 kPa组、100 kPa组,用自制的反馈式气控压应力细胞培养装置分别持续加压(对照组不予加压),于加压后1、2、3、4d,分别做葡萄糖转移实验,同时取加压4d后的脂肪颗粒做四甲基偶氮噻唑蓝(MTT)实验以及组织学HE染色,统计学分析比较各组脂肪颗粒活性的大小.结果 脂肪颗粒经不同大小压应力作用后,其转移葡萄糖的量随着压应力的增加而减少(P＜0.01),并且这种影响具有时间依赖性;MTT实验结果显示脂肪颗粒的吸光度值(A492nm)随压应力增 加而减少(P＜0.05),且与葡萄糖转移实验相关(r=0.838,P＜0.01);组织学切片提示脂肪细胞损伤率随压应力增加而增大(P＜0.01).结论 压应力会损伤脂肪颗粒的活性,建议在自体脂肪移植的临床应用中,在受区分离开阔的空间,以尽量减少压应力对脂肪颗粒活性的损伤.     

Cryopreservation of human semen

A review is given of the techniques for the cryopreservation of human semen, including the preparation of cryoprotective media, the use of ampoules, straws, and pellets, and freezing and thawing techniques. The use of cryopreserved semen for therapeutic artificial insemination by donor is described. The advantages of cryopreserved semen over fresh donor semen mostly lie in the ability to exclude infections before use and the extra convenience, in spite of the lower success rate and increased cost. The recovery of sperm motility on thawing is described, as are other methods for assessing the degree of damage to the spermatozoa by the freezing procedure. The success rates reported by large semen banks are summarized.     

Function and viability of human islets encapsulated in alginate sheets: in vitro and in vivo culture

Islet encapsulation offers an immune system barrier for islet transplantation, and encapsulation within an alginate sheetlike structure offers the ability to be retrievable after transplanted. This study aims to show that human islets encapsulated into islet sheets remain functional and viable after 8 weeks in culture or when transplanted into the subcutaneous space of rats. Human islets were isolated from cadaveric organs. Dissociation and purification were done using enzymatic digestion and a continuous Ficoll-UWD gradient. Purified human islets were encapsulated in alginate sheets. Human Islet sheets were either kept in culture, at 37°C and 5% CO₂, or transplanted subcutaneously into Lewis rats. After 1, 2, 4, and 8 weeks, the human islet sheets were retrieved from the rats and assessed. The viability of the sheets was measured using fluorescein diacetate (FDA)/propidium iodide (PI), and function was measured through glucose-stimulated insulin release, in which the sheets were incubated for an hour in low-glucose concentration (2.8 mmol/L) and then high (28 mmol/L), then high (28 mmol/L) plus 3-isobutyl-1-methylxanthine (50 μm). Human islet sheets remained both viable, above 70%, and functional, with a stimulation index (insulin secretion in high glucose divided by insulin secretion in low glucose) above 1.5, over 8 weeks of culture or subcutaneous transplantation. Islet transplantation continues to make advances in the treatment of type 1 diabetes. These preliminary results suggest that encapsulated islets sheets can survive and maintain islet viability and function in vivo, within the subcutaneous region.     

Mature adipocytes inhibit in vitro differentiation of human preadipocytes via angiotensin type 1 receptors

Recent studies suggest that angiotensin II (Ang II) plays a role in the adipogenesis of murine preadipocytes. Here, we examined the role of Ang II for the differentiation of primary cultured human preadipocytes. Preadipocytes were isolated from human adipose tissue and stimulated to differentiate. Quantitation of gene expression during adipogenesis was performed for renin-angiotensin system (RAS) genes. The influence of the RAS on adipogenic differentiation was investigated by addition of either angiotensinogen (AGT), Ang II, or angiotensin receptor antagonists to the differentiation medium. We also examined the influence of adipocytes on adipogenesis by co-culture experiments. Expression of the RAS genes AGT, renin, angiotensin-converting enzyme, and Ang II type 1 receptor increased during adipogenesis. Stimulation of the Ang II type 1 receptor by Ang II reduced adipose conversion, whereas blockade of this receptor markedly enhanced adipogenesis. Adipocytes were able to inhibit preadipocyte differentiation in the co-culture, and this effect was abolished by blockade of the Ang II type 1 receptor. This finding points to a functional role of the RAS in the differentiation of human adipose tissue. Because AGT secretion and Ang II generation are characteristic features of adipogenesis, we postulate a paracrine negative-feedback loop that inhibits further recruitment of preadipocytes by maturing adipocytes.     

Omeprazole does not alter human sperm motility,viability or DNA integrity in vitro

The effect of omeprazole, a commonly used drug belongs to proton–‐pump inhibitor class, on human sperm function is still undetermined. Here, we hypothesised that addition of omeprazole to the ejaculated human semen may affect sperm parameters, and hence sperm function. Therefore, we assessed the in vitro effect of omeprazole on human sperm motility, viability and DNA integrity. Sixty‐six normozoospermic semen samples were collected randomly from men who attended the andrology laboratory at King Abdullah University Hospital. Sperm motility, viability and DNA breaks were assessed in the presence (1‐hr incubation at 37°C) of omeprazole at 5, 10, 20 and 50 µM compared to control (0 µM). None of the examined sperm parameters, at any tested omeprazole concentration, showed significant difference (p > 0.05) compared with the control. In conclusion, omeprazole at 5, 10, 20 and 50 µM does not alter human sperm motility, viability or DNA integrity in vitro.     

海藻糖对低温保存皮肤的作用

目的 研究非渗透性低温保护剂海藻糖联合应用二甲基亚砜(DMSO)，用于人类皮肤的低温储存，并与皮厍常规应用的二甲基亚砜(DMSO)、丙二醇(propyleneglycol)、DMEM进行比较，以寻求更佳的皮肤冷冻保护剂。方法将新鲜成人皮肤分为5组，液氮冻存7、14d后复温，采用病理形态学、酶组织化学、组织代谢等方法，分别对海藻糖／二甲基亚砜、二甲基亚砜／丙二醇、二甲基亚砜／无血清角质细胞培养基(KSFM)、DMEM作为冷冻保护剂保存后的皮肤活力进行比较。结果O．5mol／L海藻糖／二甲基亚砜作为保护剂所冷冻的皮肤，复温后其活性明显高于其他保护荆组。结论海藻糖与二甲基亚砜联合应用是较佳的冷冻保护剂，皮库在常规应用保护剂时可以在渗透性保护剂中加入海藻糖。     

成年猪半月板体外器官培养模型的建立

[目的]观察成年猪半月板在体外器官培养条件下的组织变化特点,为在体外器官培养下进行半月板研究提供实验依据.[方法]成年猪8头,取双膝内、外侧半月板,从每个半月板体部横行切取宽约8 mm标本1份,共32份标本,进行体外器官培养.分别于培养0、2、4、6周后随机取出8个标本行组织学检查,HE染色:观察半月板内侧1/3区域,计数每个视野的细胞数,比较各时间点间的差异;番红O染色:观察半月板内侧1/3区域,记录染色程度,半定量评分,比较各时间点间的差异.[结果]培养0、2、4、6周后,各时间点间每高倍视野的细胞数有显著性差异(P＜0.05),两两比较均有统计学差异(P＜0.05).培养0、2、4、6周后,各时间点间番红O染色半定量评分有统计学差异(P＜0.05),两两比较均有统计学差异(P＜0.05).[结论]在体外器官培养模型中进行半月板的短期研究是可行的,随培养时间延长,半月板软骨细胞密度及活性逐渐下降.     

人输卵管上皮细胞培养液对人精子活力的影响

哺乳动物输卵管是精子获能、受精和早期胚胎发育的重要场所，并通过其分泌的物质协助完成上述过程。为了解其对精子活力的影响，我们观察了月经中期人输卵管上皮细胞培养液和管腔液对人精子活动率、平均运动速度、高活力精子比例及活动指数的影响。结果在共育不同时间后对上述指标均有显著促进作用，输卵管上皮细胞培养液的作用强于管腔液。说明人输卵管上皮细胞培养液可提高人精子活动能力和生存能力。     

人脂肪细胞的可塑性

目的 探索体外培养环境下人成熟脂肪细胞的去分化现象,旨在挖掘其作为种子细胞的潜能,为组织工程研究开辟新思路.方法 自成人吸脂术后抽吸物提取成熟脂肪细胞及脂肪组织来源干细胞(adipose-derived stromal cells,ASCs),天花板贴壁培养法诱导成熟脂肪细胞去分化,观察细胞形态变化,获得去分化脂肪细胞(dedifferentiated adipocytes,DA).相同的条件下,MTT比色法比较DA、ASCs活性并绘制细胞生长曲线;流式细胞仪鉴定DA、ASCs表面分子的表达;油红O染色、茜素红染色、阿尔辛蓝染色分别鉴定DA、ASCs成脂分化、成骨、成软骨分化能力.结果 人成熟脂肪细胞在体外培养环境下能去分化为成纤维细胞状DA;MTT比色法测细胞活性:DA、ASCs均有很强的增殖能力,两者差异无统计学意义;流式细胞仪测定:DA、ASCs中HLA-ABC、CD29、CD44均为阳性,CD45、CD34、CD106均为阴性;成脂分化2周,油红O染色可见DA、ASCs内出现红色脂滴;成骨分化2周,茜素红染色可见DA、ASCs内红色钙盐沉积;成软骨分化2周,阿尔辛蓝染色可见DA、ASCs内软骨基质沉积.结论 成熟的脂肪细胞在体外培养条件下可成为DA,DA具有很强的增殖活性,表达部分干细胞特征性表面蛋白,有成骨、成软骨及强大的成脂分化能力,有望成为组织工程优秀的种子细胞.

Abstract：

Objective To explore the dedifferentiation phenomenon of human mature adipocytes cultured in vitro and to discuss the possibility of using dedifferentiation adipocytes ( DA ) as seed cells.Methods Mature adipocytes and ASCs were harvested from human fat aspirates. Mature adipocytes were cultured and induced to DA by ceiling adherent culture method. Cell morphology were observed during the whole process. Viabilities of DA and ASCs were compared by MTT chromatometry and cell growth curves were drawn based on it. Cell surface markers of DA and ASCs were detected by flow cytometry. The adipogenic,osteogenic and chondrogenic ability of DA and ASCs were assessed by oil red O staining,alizarin bordeaux staining and alcian blue staining, respectively. Regults Human mature adipocytes can dedifferentiate into fibroblast-shaped DA. MTT chromatometry assay demonstrated that DA and ASCs both had strong reproductive activity, with no significant difference between them. Flow cytometry assay demonstrated that both DA and ASCs expressed HLA-ABC, CD29 and CD44, while didn't express CD45,CD34 and CD106. After two weeks of adipogenic differentiation, lipid droplets could be displayed by oil red O staining in both DA and ASCs. After two weeks of osteogenic differentiation, calcium salts mineralization in DA and ASCs could be detected by alizarin bordeaux staining. After two weeks of chondrogenic differentiation, matrix of cartilage cells in DA and ASCs could be detected by alcian blue staining. Conclusions Mature adipocytes can be dedifferentiated into DA in vitro. DA has strong reproductive activity, as well as osteogenic, chondrogenic ability and strong adipogenic ability. It expresses some of the stem cell-related cell surface proteins and is a promising seed cell for adipose tissue engineering.     

The cellular plasticity of human adipocytes

Little is known regarding the biology of fat considering its extensive use clinically in soft tissue implantation. Free-fat transfer is problematic the result of graft site volume loss, appearing histologically as the replacement of mature adipocytes with a fibroblast-like infiltrate. We hypothesize that these histologic changes reflect a dedifferentiation of ischemic mature adipocytes instead of, or in addition to, a more traditional response. To explore this hypothesis, we studied the in vitro morphologic changes of cultured mature human adipocytes isolated from liposuctioned adipose tissue. Most adipocytes over time lost significant amounts of intracellular lipid. Ultimately, these cells lost all lipid, appeared fibroblastic, and proliferated to confluence. Adipogenic induction of such dedifferentiated adipocytes resulted in reaccumulation of intracellular lipid. This study demonstrates that mature adipocytes can be cultured from human liposuctioned fat, they can dedifferentiate into fibroblastic cells, and the fibroblast-like cells can be expanded and turned into lipid-synthesizing adipocytes. Exploration of this cellular plasticity might ultimately yield important insights into free-fat transfer and novel tissue-engineering strategies.     

Quantitative measurement of patellofemoral joint stability: force-displacement behavior of the human patella in vitro.

Patellofemoral joint instability is a common clinical problem. However, little quantitative data are available describing the stability characteristics of this joint. We measured the stability of the patella against both lateral and medial displacements across a range of knee flexion angles while the quadriceps were loaded physiologically. For eight fresh-frozen knee specimens a materials testing machine was used to displace the patella 10 mm laterally and 10 mm medially while measuring the required force, with 175 N quadriceps tension. The patella was connected via a ball-bearing patellar mounting 10 mm deep to the anterior surface to allow natural tilt and other rotations. Patellar force-displacement behavior was tested at flexion angles of 0 degrees, 10 degrees, 20 degrees, 30 degrees, 45 degrees, 60 degrees, and 90 degrees. Significant differences were found between the lateral and medial restraining forces at 10 mm displacement. For lateral displacement, the restraining force was least at 20 degrees of knee flexion (74 N at 10 mm displacement), rising to 125 N at 0 degrees and 90 degrees of knee flexion. The restraining force increased progressively with knee flexion for medial patellar displacement, from 147 N at 0 degrees to 238 N at 90 degrees. With quadriceps tension, the patella was more resistant to medial than lateral displacement. Our finding that lateral patellar displacement occurred at the lowest restraining force when the knee was flexed 20 degrees agrees with clinical experience of patellar instability.