In vitro isolation and cultivation of rabbit tracheal epithelial cells using tissue explant technique

Epithelial cells from tracheal mucosa offer significant potential as a cell source in development of tissue-engineered trachea. The purpose of this study was to investigate and optimize a suitable culture system for tracheal epithelial cells, including the methods of primary culture, passage, identification, and cryopreservation. Epithelial cells were isolated from rabbit tracheal mucosa using tissue explant technique and were subjected to immunohistochemistry, immunofluorescence, and cryopreservation after purification. Epithelial cells reached confluency at 14–15 d. Immunohistochemical staining for cytokeratin showed brown yellow-positive cytoplasm and blue-counterstained nuclei, while immunofluorescence staining for cytokeratin showed green-positive cytoplasm and clear cell outline, indicating that the cultured cells had properties of epithelial cells. After recovery, epithelial cells exhibited high survival and viability. The results demonstrated that in vitro isolation and cultivation model was successfully established to provide high proliferative capacity, typical morphology and characteristics of tracheal epithelial cells from trachea mucosa by the use of the tissue explant technique.     

Serum-free cryopreservation of porcine hepatocytes

The use of porcine hepatocytes in xenotransplantation, bioartificial liver support or pharmacological approaches demands serum-free cryopreservation protocols yielding high quality, viable, functional hepatocytes. Here, primary porcine hepatocytes were frozen without serum in liquid nitrogen by the use of a computer-assisted freezing device. After thawing, more than 90% of the initial hepatocytes were lost, in part because of damage to genomic DNA. When cryoprotectants were used, the loss was lowered to 70% of the initial cell number; 90% of the remaining cells excluded trypan blue indicating a high degree of viability. Cells were seeded serum-free onto collagen-coated plastic dishes to determine proliferation and retainment of specific functions representing prominent features of hepatocytes in vivo. Whereas no cells adhered to the substratum effectively in conventional culture medium, the addition of conditioned medium derived from hepatic non-parenchymal cells improved attachment. Cells proliferated, retained hepatocyte-specific functions, such as urea production and cytochrome P450 activity, and expressed liver-specific genes to levels observed in non-cryopreserved hepatocytes. Thus, serum-free cryopreserved primary porcine hepatocytes may serve as a valid source of cells for downstream applications. The cells seem to function adequately when an appropriate environment is chosen for recovery after cryopreservation, an ultimate demand for the clinical application of human hepatocytes.     

Interaction between hepatocytes and collagen gel in hollow fibers

Gel entrapment culture of primary mammalian cells within collagen gel is one important configuration for construction of bioartificial organ as well as in vitro model for predicting drug situation in vivo. Gel contraction in entrapment culture, resulting from cell-mediated reorganization of the extracellular matrix, was commonly used to estimate cell viability. However, the exact influence of gel contraction on cell activities has rarely been addressed. This paper investigated the gel contraction under varying culture conditions and its effect on the activities of rat hepatocyte entrapped in collagen gel within hollow fibers. The hepatocyte activities were reflected by cell viability together with liver-specific functions on urea secretion and cytochrome P450 2E1. Unexpectedly, no gel contraction occurred during gel entrapment culture of hepatocyte under a high collagen concentration, but hepatocytes still maintained cell viability and liver-specific functions at a similar level to the other cultures with normal gel contraction. It seems that cell activities are unassociated with gel contraction. Alternatively, the mass transfer resistance induced by the combined effect of collagen concentration, gel contraction and cell density could be a side effect to reduce cell activities. The findings with gel entrapment culture of hepatocytes would be also informative for the other cell culture targeting pathological studies and tissue engineering.     

Phenotype of hepatocyte spheroids in synthetic thermo-reversible extracellular matrix

Aggregates of specific cells are often regarded as a better form in artificial organs and mammalian cell bioreactors in terms of cell-specific functionality. In this study, the morphology and liver-specific functions of freshly harvested primary rat hepatocytes, which were cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were examined and compared to a control (hepatocytes in single cell form). A copolymer of N-isopropylacrylamide (98 mole % in feed) and acrylic acid (poly(NiPAAm-co-AAc)), a thermo-reversible copolymer gel matrix, was used to entrap hepatocytes either in spheroids or single cells. During a 7-day culture period, the spheroids maintained higher viability and produced albumin and urea at a relatively constant rate, while the single cell culture showed a slight increase in cell numbers and a reduction in albumin secretion. Hepatocytes cultured as spheroids present a potentially useful three-dimensional cell culture system for application in a bioartificial liver device.     

Effect of cryopreservation on the appearance and liver function of hepatocyte-like cells in cultures of cirrhotic liver of biliary atresia

Previously, we reported that non-parenchymal cell (NPC) fractions from cirrhotic liver of biliary atresia (BA) may contain stem/progenitor cells, and clusters of hepatocyte-like cells appear via hepatocyte growth factor/c-Met signaling in primary cultures of NPCs. BA is a rare and serious liver disease, and procurement of BA cells is difficult. Therefore, cryopreservation of BA liver cells is an unavoidable challenge. In this study, we examined the appearance and liver function of hepatocyte-like cells in cultures of BA liver-derived NPC fractions after cryopreservation for 1 or 6 mo using a chemically defined cryopreservation solution, STEM-CELLBANKER. Although a decrease in cell viability was observed in recovered cells after 1 mo of cryopreservation, clusters of hepatocyte-like cells appeared in the culture of cells that had been cryopreserved for 1 or 6 mo, similar to non-cryopreserved cells. In addition, these hepatocyte-like cells expressed hepatocyte-related mRNAs and demonstrated albumin production and glycogen storage. The present results suggest that hepatic stem/progenitor cells in NPC fractions may be efficiently cryopreserved, as demonstrated by the appearance of hepatocyte-like cells that show various hepatic functions even after cryopreservation. This study may lead to future BA cell therapy using the patient’s own cells.     

Cryopreservation of primary porcine liver cells in an organotypical sandwich model in a clinically relevant flat membrane bioreactor

To overcome the logistical difficulties of continuously supplying freshly-isolated, primary porcine liver cells to bioartificial liver support bioreactors, we developed a cryopreservation method using an organotypical sandwich model in a flat membrane bioreactor (FMB). We measured albumin secretion rate, urea synthesis rate and 7-ethoxy coumarin (ECOD) in long-term cultures of cryopreserved cells (up to 14 days). The albumin secretion rate was 62% that of non-cryopreserved cells at days 11 and 14. The ECOD activity was 54% that of fresh, control cells initially and increased up to 79% by the 14th day. The urea synthesis rate was stable at 60% that of the control. This study showed that cryopreserved cells can recover liver-specific functions. This result has the potential to dramatically expand the clinical application of bioartificial liver supports.     

Phenotype of hepatocyte spheroids in Arg-GLY-Asp (RGD) containing a thermo-reversible extracellular matrix

The spheroid of specific cells is often regarded as the better form in artificial organs and mammalian cell bioreactors for improved cell-specific functions. In this study, freshly harvested primary rat hepatocytes, which had been cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were examined for differentiated morphology and enhanced liver-specific functions as compared to a control set (hepatocytes in single-cell form). A copolymer of N-isopropylacrylamide (98 mole % in the feed) and acrylic acid (poly(NiPAAm-co-AAc)), and the adhesion molecule, an Arg-Gly-Asp (RGD)-incorporated thermo-reversible matrix, were used to entrap hepatocytes in the form of either spheroids or single cells. In a 28-day culture period, the spheroids in the RGD-incorporated gel maintained higher viability and produced albumin and urea at constant rates, while there was lower cell viability and less albumin secretion by the spheroids in p(NiPAAm-co-AAc). Hepatocytes cultured as spheroids in the RGD-incorporated gel would constitute a potentially useful three-dimensional cell system for application in a bio-artificial liver device.     

Enhancement of cell recovery for dissociated human embryonic stem cells after cryopreservation

Due to widespread applications of human embryonic stem (hES) cells, it is essential to establish effective protocols for cryopreservation and subsequent culture of hES cells to improve cell recovery. We have developed a new protocol for cryopreservation of dissociated hES cells and subsequent culture. We examined the effects of new formula of freezing solution containing 7.5% dimethylsulfoxide (DMSO) (v/v %) and 2.5% polyethylene glycol (PEG) (w/v %) on cell survival and recovery of hES cells after cryopreservation, and further investigated the role of the combination of Rho‐associated kinase (ROCK) inhibitor and p53 inhibitor on cell recovery during the subsequent culture. Compared with the conventional slow‐freezing method which uses 10% DMSO as a freezing solution and then cultured in the presence of ROCK inhibitor at the first day of culture, we found out that hES cell recovery was significantly enhanced by around 30 % (P < 0.05) by the new freezing solution. Moreover, at the first day of post‐thaw culture, the presence of 10 μM ROCK inhibitor (Y‐27632) and 1 μM pifithrin‐μ together further significantly improved cell recovery by around 20% (P < 0.05) either for feeder‐dependent or feeder‐independent culture. hES cells remained their undifferentiated status after using this novel protocol for cryopreservation and subsequent culture. Furthermore, this protocol is a scalable cryopreservation method for handling large quantities of hES cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Is axenicity crucial to cryopreserve microalgae?

Large culture collections of microalgae and cyanobacteria such as the Coimbra Collection of Algae (ACOI) hold unialgal cultures consisting of a population of cells/colonies of a certain species. These cultures are usually non-axenic, as other organisms such as bacteria and microfungi are also present in culture due to co-isolation. Attention has been recently given to partner organisms since studies indicate that some bacteria are important for nutrient uptake of the algal cells, acting as simbionts. Despite this benign effect in the actively growing cultures, when cryopreservation is applied for inactive-stage storage, these organisms may recover faster than the algae, thus affecting their recovery and the viability assessments. In this study, a set of mucilaginous ACOI microalgae were selected, cell features known for their relevance in cryopreservation success were recorded and simple two-step cryopreservation tests were applied. Thawed samples were transferred to fresh culture medium for recovery. Viability was assessed and partner organism proliferation (pop) was recorded. Results were analyzed by t-tests. Statistical models allowed us to support the known tendency for small, unicellular algae with no outer structures to be successfully cryopreserved and the negative effect of vacuoles in the cell prior to cryopreservation. On average cryopreservation with MeOH or Me₂SO led to the recovery of nearly half the cells. It was found that the cryoprotection step with MeOH is when pop is triggered and that the use of Me₂SO can prevent this effect. Progress on understanding the cultured consortia will assist the improvement of cryopreservation and research using microalgal cultures.     

A novel full-scale flat membrane bioreactor utilizing porcine hepatocytes: cell viability and tissue-specific functions

When designing an extracorporeal hybrid liver support device, special attention should be paid to providing the architectural basis for reconstructing a proper cellular microenvironment that ensures highest and prolonged functional activity of the liver cells. The common goal is to achieve high cell density culture and to design the bioreactor for full-scale primary liver cell cultures under adequate mass transfer conditions. An important aim of this study was to evaluate the biochemical performance of a flat membrane bioreactor that permits high-density hepatocyte culture and simultaneously to culture cells under sufficient oxygenation availability conditions comparable to the in vivo-like microenvironment. In such a bioreactor pig liver cells were cultured within an extracellular matrix between oxygen-permeable flat-sheet membranes. In this investigation we used a novel scaled-up prototype consisting of up to 20 modules in a parallel mode. Each module was seeded with 2 x 10(8) cells. Microscopic examination of the hepatocytes revealed morphological characteristics as found in vivo. Cell concentration increased in the first days of culture, as indicated by DNA measurements. The performance of the bioreactor was monitored for 18 days in terms of albumin synthesis, urea synthesis, ammonia elimination, and diazepam metabolism. The ability of the hepatocytes to synthesize albumin and urea increased during the first days of culture. Higher rates of albumin synthesis were obtained at day 9 and remained at a value of 1.41 pg/h/cell until day 18 of culture. The rate of urea synthesis increased from 23 ng/h/cell to 28 ng/h/cell and then remained constant. Cells eliminated ammonia at a rate of about 56 pg/h/cell, which was constant over the experimental period. Hepatocytes in the bioreactor metabolized diazepam and generated three different metabolites: nordiazepam, temazepam, and oxazepam. The production of such metabolites was sustained until 18 days of culture. These results demonstrated that the scale-up of the bioreactor was assessed, and it could be demonstrated that the device design aimed at the reconstruction of the liver-specific tissue architecture supported the expression of liver-specific functions of primary pig liver cells.     

美洲鲥雄性生殖细胞冷冻保存及移植

采用分离细胞冻存和组织块直接冻存2种方法, 进行美洲鲥(American shad, Alosa sapidissima)精巢细胞的长时间冷冻保存(>250d), 并比较分析2种不同冻存方法对美洲鲥雄性生殖细胞的冻存效果。解冻复苏后用Hochest33342和PI共染细胞核, 分析统计各期雄性生殖细胞的存活率, 结果显示组织块冻存方法所得精原干细胞和精母细胞的存活率明显高于分离细胞冻存的; 而精细胞及其他细胞存活率在2种方法间无显著差异; 特别是, 镜检发现组织块冻存方法所得精子存活率高达93.83%, 说明此冻存方法能同时高效地冻存美洲鲥各期生殖细胞, 包括成熟的精子。同时, 将组织块冻存的美洲鲥生殖细胞用PKH26染色标记后移植到出苗第1天的斑马鱼仔鱼中, 在细胞植入后5d仍能在受体中检测到供体细胞, 且有部分供体细胞能与内源生殖细胞共定位, 表明经过长时间冷冻保存的美洲鲥生殖细胞仍具有生殖细胞特性, 且能整合到斑马鱼受体性腺原基。研究结果为进一步开展美洲鲥, 或其他洄游性鱼类的生殖细胞发育、培养及种质资源保存等研究工作奠定了技术理论基础。     

Comparison of stem cell viability of bone marrow cryopreserved by two different methods

Two different cryogenic methods were used to study the preservation of murine bone marrow cells. Compared to the classical methods, in which separated mononuclear marrow cells in 10% dimethyl sulfoxide (DMSO) were cryopreserved in liquid nitrogen (-196 degrees C), a modified technique was carried out by cryopreservation of unfractionated marrow cells in a mixed protectant of 5% DMSO and 6% hydroxyethyl starch (HES) at -80 degrees C. Samples that were separately thawed after storage for 1, 4, 8, and 12 weeks were assayed for cell viability and recovery of CFU-GM and CFU-S. No macroscopic clumping of cells was noted either in fractionated or in unfractionated marrow cell cryopreservations. A mild damage, about 25% reduction of stem cells, was found at 1 week and did not deepen further. It seems that the greatest loss of stem cells occurred in the process of cryopreservation itself. Compared to prefreeze values, both a high number of cells that excluded trypan blue (87 +/- 3.4%) and a high recovery of CFU-GM (75 +/- 9.8%) and CFU-S (74 +/- 11.2) were observed in unfractionated marrow samples cryopreserved with the DMSO/HES mixture at -80 degrees C for 3 months and these results were very similar to those obtained from fractionated mononuclear marrow cells cryopreserved at -196 degrees C. The DMSO/HES protectant provides a simplified bone marrow cryopreservation technique that should be favorable to clinical application because of its high stem cell recovery and avoidance of cell-separation manipulation.     

Improved long-term culture of hepatocytes in a hydrogel containing Arg-Gly-Asp (RGD)

Freshly harvested primary rat hepatocytes, which had been entrapped in a synthetic extracellular matrix, were examined for differentiated morphology and enhanced liver-specific functions for long-term culture. A copolymer of N-isopropylacrylamide (98 mol % in the feed) and acrylic acid [poly(NiPAAm-co-AAc)], and the adhesion molecule, Arg-Gly-Asp (RGD), incorporated into a hydrogel, were used to entrap hepatocytes. Over 28 days' culture, the hepatocytes in the RGD-incorporated gel maintained higher viability and produced albumin and urea at constant rates, while there was lower cell viability and less albumin secretion by hepatocytes in poly(NiPAAm-co-AAc). Hepatocytes cultured in the gel with RGD incorporated into it constitutes a potentially useful three-dimensional cell system for application in a bio-artificial liver device.     

Impact of Procedural Steps and Cryopreservation Agents in the Cryopreservation of Chlorophyte Microalgae

The maintenance of traditional microalgae collections based on liquid and solid media is labour intensive, costly and subject to contamination and genetic drift. Cryopreservation is therefore the method of choice for the maintenance of microalgae culture collections, but success is limited for many species. Although the mechanisms underlying cryopreservation are understood in general, many technical variations are present in the literature and the impact of these are not always elaborated. This study describes two-step cryopreservation processes in which 3 microalgae strains representing different cell sizes were subjected to various experimental approaches to cryopreservation, the aim being to investigate mechanistic factors affecting cell viability. Sucrose and dimethyl sulfoxide (DMSO) were used as cryoprotectants. They were found to have a synergistic effect in the recovery of cryopreserved samples of many algal strains, with 6.5% being the optimum DMSO concentration. The effect of sucrose was shown to be due to improved cell survival and recovery after thawing by comparing the effect of sucrose on cell viability before or after cryopreservation. Additional factors with a beneficial effect on recovery were the elimination of centrifugation steps (minimizing cell damage), the reduction of cell concentration (which is proposed to reduce the generation of toxic cell wall components) and the use of low light levels during the recovery phase (proposed to reduce photooxidative damage). The use of the best conditions for each of these variables yielded an improved protocol which allowed the recovery and subsequent improved culture viability of a further 16 randomly chosen microalgae strains. These isolates included species from Chlorellaceae, Palmellaceae, Tetrasporaceae, Palmellopsis, Scenedesmaceae and Chlamydomonadaceae that differed greatly in cell diameter (3–50 µm), a variable that can affect cryopreservation success. The collective improvement of each of these parameters yielded a cryopreservation protocol that can be applied to a broad range of microalgae.     

A novel human hepatoma cell line, FLC-4, exhibits highly enhanced liver differentiation functions through the three-dimensional cell shape

We characterized three-dimensional human hepatoma cell lines, functional liver cell (FLC) cell lines, to establish a highly differentiated hepatoma cell line. We investigated the effect of extracellular matrix and cell morphology on liver-specific gene expression in FLC cells. The hepatocyte nuclear factor-4α (HNF-4α) and other liver-specific gene expressions were enhanced in spherical FLC-4 cells on EHS-gel, but other human hepatoma cells such as HepG2 did not show the enhancement. Importantly, the liver-specific gene expression levels in spherical FLC-4 cells cultured on EHS-gel were comparable to those of human liver and were much higher than those of other human hepatoma cell lines. The major matrix components and growth factors in EHS-gel did not affect cell shape and liver functions. To exclude any effect of the extracellular matrix, we made spherical FLC-4 cells by actin filament disruption. The actin-disrupted spherical cells also showed an enhanced liver-specific gene expression. We concluded that three-dimensional cell shape per se is one of the most important determinants of liver differentiation functions in FLC-4 cells. Cell morphology-dependent induction of liver-specific gene expression was mediated through microtubule organization. In conclusion, differentiation of FLC-4 human hepatoma cell line can be enhanced to a human liver-like level through the three-dimensional cell shape in a microtubule-dependent manner.     

海藻酸微囊在细胞冷冻保存中的研究现状及应用

细胞的冷冻保存是细胞生物学实验中重要的实验技术.长期以来,人们使用冷冻保存液重悬细胞后进行冷冻储存,但是近年来,众多研究者发现传统冷冻方案往往会导致细胞活率大幅下降和细胞功能方面受损,从而很难满足生物医学、组织再生工程、细胞移植技术等高新技术的要求.所以研究者提出利用三维海藻酸微囊包埋细胞后再进行冷冻保存,从而在保证较高细胞活率的同时维持细胞的原有功能,有效的提高细胞的冷冻保存效率.本文概述了海藻酸微囊在细胞冷冻保存过程中的研究现状,同时对其应用进行了展望,以期为后续研究工作提供参考.     

Spheroid formation and expression of liver-specific functions of human hepatocellular carcinoma-derived FLC-4 cells cultured in lactose-silk fibroin conjugate sponges

This study presents a hepatic tissue engineering application of three-dimensional (3D) porous sponges composed of lactose-silk fibroin (SF) conjugates (Lac-CY-SF) bearing β-galactose residues, hepatocyte-specific ligands. Lac-CY-SF sponges were prepared by freeze-drying, followed by immersion in a series of methanol aqueous solutions. Lac-CY-SF sponges showed heterogeneous pore structure with round pores about 100 μm in diameter and elongated pores 250-450 μm in length and 100-150 μm in breadth. To employ a 3D Lac-CY-SF culture system, human hepatocellular carcinoma-derived FLC-4 cells were seeded in Lac-CY-SF sponges and cultured up to 3 weeks. FLC-4 cell culture in collagen and SF sponges was also performed for comparison with the cell response to Lac-CY-SF sponges. Within 5 days of culture, FLC-4 cells cultured in Lac-CY-SF sponges, as well as the cells cultured in collagen sponges, formed multicellular spheroids with diameters from 30 to 100 μm more efficiently than did the cells cultured in SF sponges. After 3 weeks of culture, WST-1 viability assay revealed that shrinkage suppression of Lac-CY-SF sponges enabled the maintenance of viable FLC-4 cells for a long time, while the shrinkage and disintegration of collagen sponges prevented the maintenance of the cells. FLC-4 cells cultured in Lac-CY-SF sponges exhibited greater elevation of albumin secretion and sustained a higher albumin level compared with the cells cultured in collagen and SF sponges during the 3 week cultivation period. FLC-4 cells cultured in Lac-CY-SF sponges for 3 weeks expressed genes related to liver-specific functions such as transferrin and HNF-4α. On the other hand, the cells cultured in collagen and SF sponges for 3 weeks did not express these genes. These results indicated the very promising properties of Lac-CY-SF sponges as a scaffold for long-term culture of functional FLC-4 cells to study drug toxicity and hepatocyte metabolism in humans and develop a bioartificial liver model.     

Role of apoptotic signaling pathway in metabolic disturbances occurring in liver tissue after cryopreservation: Study on rat precision-cut liver slices

Precision-cut liver slices in culture (PCLS) appears as a useful and widely used model for metabolic studies; the interest to develop an adequate cryopreservation procedure, which would allow maintaining cell integrity upon incubation, is needed to extend its use for human tissues. We have previously shown that cryopreservation of rat PCLS leads to caspase-3 activation and early alterations of their K+ content upon incubation. In this study, we tested the hypothesis that counteracting intracellular K+ loss and/or interfering with cell death signaling pathways could improve the viability of cryopreserved PCLS. PCLS were prepared from male Wistar rat liver and cryopreserved by rapid freezing before incubation. The addition of a caspase inhibitor-Z-DEVD-FMK (2.5 microM)-in the culture medium did not improve viability of cryopreserved PCLS. Incubation of cryopreserved PCLS in a K+ rich medium (135 mM) increased K+ content and avoided caspase-3 activation, but did not improve cell viability. Caspase-3 inhibition, a decrease in cell lysis, and improvement of glycogen content were observed in cryopreserved PCLS after addition of LiCl (100 mM) in the incubation medium. These results indicate that, even if caspase-3 activation is linked to the K+ loss in cryopreserved PCLS, its inhibition does not allow restoring the metabolic capacities. LiCl, acting on a target upstream of caspase-3 inhibition, improves cell viability and allows glycogen accumulation when added in culture medium of cryopreserved PCLS; and could thus be considered as an interesting adjuvant in the culture of cryopreserved PCLS.