Design of a fluidized bed bioartificial liver

The aim of this study was to design a bioreactor for extracorporeal liver supply containing alginate beads in a fluidized bed regimen. The objective was to achieve a satisfactory mixing into the bioreactor to promote the potential exchanges and mass transfers. First, we checked whether both present phases (solid: alginate beads; liquid: saline solution at 20 degrees C) might allow for this fluidization. Then the optimal design was defined as a function of the required operating conditions, bead volume, and perfusion flow rate; the bioreactor cross section and height especially needed to be adjusted. The efficient fluidization, under optimized conditions, was proven through the follow-up of the head losses generated by the fluidized bed. Criteria for scaling up were also determined.     

Towards the development of a bioartificial pancreas: Effects of poly-l-lysine on alginate beads with BTC3 cells

A bioartificial tissue construct that consists of insulin-secreting cells entrapped in an alginate/poly-l-lysine (PLL) matrix offers a promising approach for the treatment of type I diabetes. Use of transformed cells has been proposed as a solution to the cell availability problem posed by islets. The growth characteristics of transformed cells in their sequestered environment and the effects of PLL on their metabolic and secretory activities have not yet been characterized. Our data demonstrate that mouse insulinoma βTC3 cells proliferate while they are entrapped in both PLL-free and PLL-coated alginate beads. During this process, cell aggregates develop in the bead periphery, which increase in number and size with time. PLL is crucial for the long-term in vitro structural stability of beads, and it does not appear to affect the metabolic and secretory activities of entrapped βTC3 cells. The implications of these findings in the development of a bioartificial pancreatic construct based on transformed cells are discussed.     

以海藻酸三维支架构建骨髓间充质干细胞源性生物人工肝组织的实验研究

目的 以骨髓间质干细胞(bone mesenchymal stem cell,BMSC)为种子细胞,以海藻酸三维支架为载体,研究两者的相容性及其构建生物人工肝组织的可能性.方法 将大鼠BMSC接种于海藻酸三维支架上,采用倒置相差显微镜、扫描电镜分别检测BMSC的黏附、生长与增殖情况,评价两者的相容性;同时以表皮生长因子(EGF)、肝细胞生长因子(HGF)、成纤维生长因子4(FGF-4)等细胞因子混合液诱导BMSC向肝细胞分化,检测培养液内尿素氮和白蛋白含量,RT-PCR检测培养细胞ALB和AFP基因的表达,糖原染色及免疫荧光法检测细胞糖原合成功能和CK-18的表达. 结果大鼠BMSC在海藻酸三维支架上能正常地黏附、生长和增殖;于海藻酸三维支架内加入诱导液作用于BMSC后,培养液内尿素和ALB的含量逐渐上升,RT-PCR检测到培养细胞有AFP和ALB基因的表达,同时被诱导的细胞具有糖原储存的功能并表达CK-18.结论 海藻酸三维支架与大鼠BMSC具有良好的相容性;以EGF、HGF、FGF-4等细胞因子混合液为诱导因子,能在海藻酸三维支架上成功诱导BMSC向肝细胞分化;海藻酸三维支架和BMSC可望成为肝脏组织工程理想的种子细胞和细胞载体.     

Comparison of porcine hepatocytes with human hepatoma (C3A) cells for use in a bioartificial liver support system

Cells from primary porcine hepatocytes (PPH) and the immortalized human hepatoma cell line C3A are both used in bioartificial liver support systems (BALSS). In this work the viability and metabolic capacity of PPH and C3A cells cultured in different media were compared. Also, because the cells come into direct or indirect contact with human blood components in BALSS, the effects of human complement on survival and functions of the cells was evaluated. For short-term culture, maintenance of PPH viability was essential for retention of P450IA1 activity (r = 0.882, p < 0.01) and effective ammonia clearance (r = −0.791, p < 0.01). When cell viability was below 60% P450IA1 activity could not be recorded and nitrogen elimination activity significantly diminished. In contrast to PPH, ammonia levels were markedly increased for C3A cells in all culture media tested (p < 0.01). Ammonia increase correlated with C3A viability (r = 0.896, p < 0.05). PPH metabolic function was superior to that of the C3A cell line when evaluated by P450IA1 activity, ammonia removal, and amino acid metabolism. When PPH were incubated in human plasma (HP) or human serum (HS) there was rapid and irreversible deterioration of viability occurring within 9 h. This toxic effect could be prevented by the inactivation of complement. When sodium citrate dissolved in dextrose was added to medium, there was considerable damage to both PPH and the C3A cell line. However, there was no demonstrable toxic effect when hepatic cells of either type were exposed to heparin. We conclude that PPH cultivated in complement-inactivated HP or HS are to be preferred to C3A for clinical application of BALSS, and that heparin should be preferred for anticoagulation in BALSS.     

Culture and characterization of human hepatocytes obtained after graft reduction for liver transplantation: a reliable source of cells for a bioartificial liver

This article describes results obtained when human liver cells obtained from reduced grafts are cultured in a chemically defined medium. Remnants of livers after reduction for pediatric transplantation were processed by a multiple cannulation system through the existing vasculature, which allowed the homogeneous perfusion of collagenase. The graft weight ranged between 55 and 1000 g (median value: 145.6 g). The yield ranged between 0.13 x 10(6) and 38 x 10(6) cells/g of tissue (median value 14.73 x 10(6) cells/g), and the viability was 61.17 +/- 27.43%. The total number of cells ranged between 57.6 x 10(6) and 12 150 x 10(6) cells (median value: 740 x 10(6) cells). Cells were cultured for 30 days. Albumin synthesis was observed during the first 2 weeks, with a peak value at day 6 (27.85 +/- 1.77 micro g/mL). Urea production was detected during the first week (peak value at day 6: 17.12 +/- 2.11 mg/dL). Light microscopy showed the presence of cells in a monolayer. Biliary pigments were observed at day 20. By immunohistochemistry, positive cells for albumin, for hepatocyte marker, cytokeratin 19, CD 34, CD 68, and for alpha actin for smooth muscle, were observed. Our results showed that hepatocytes obtained from reduced liver grafts are easily cultured and are able to maintain viability and functionality in vitro. This alternative source of human cells maintained under controlled culture conditions may play an important role in the development of a bioartificial liver.     

间充质干细胞在藻酸盐微球溶液中增殖和分化的实验研究

[目的]探讨藻酸盐微球作为缓释材料在骨组织工程中对种子细胞的增殖和分化的影响。[方法]制成不同浓度的藻酸盐微球溶液，将间充质干细胞接种于其中，观察其生长和增殖情况；置换成骨诱导培养基后，通过检测细胞内碱性磷酸酶的量以及细胞外钙基质分泌来评价间充质干细胞的分化情况。[结果]不同浓度的藻酸盐微球溶液对间充质干细胞的增殖影响不同，随着浓度的增高间充质干细胞的增殖越受抑制，浓度达到10g／L时不适合细胞生长；在成骨诱导培养基中，藻酸盐可以促进细胞内碱性磷酸酶合成量的增加，并表现剂量依赖性，即藻酸盐浓度越高效应越明显，生长在藻酸盐溶液中的间充质干细胞分化为成骨细胞并分泌细胞外钙基质。[结论]一定剂量的藻酸盐微球溶液对间充质干细胞的增殖影响很小，且可促进细胞内成骨活性分子的合成，适于作为骨组织工程的缓释材料。     

生物人工肝中细胞材料的研究进展

急性肝功能衰竭是一类死亡率极高的综合症候群.近年来,虽然临床治疗手段不断改进,死亡率仍然高达50%～80%,暴发性肝功能衰竭者甚至町达90%以上[1].     

生物人工肾小管上皮种子细胞的建立

目的 利用人Nanog基因转染生物人工肾的种子细胞（肾近曲小管上皮细胞HKC），提高其增殖能力，在较短时间内获得大量种子细胞，为克服肾脏组织工程种子细胞来源的匮乏及生物人工肾的快速构建提供新途径。 方法 制备含有人Nanog基因的复制缺陷型重组腺相关病毒血清2型病毒颗粒rAAV2-hNanog，用rAAV2-hNanog转染肾小管上皮细胞,然后采用RT-PCR检测hNanog基因在转染的肾小管上皮细胞中的表达，再用MTT、免疫荧光及激光共聚焦技术检测hNanog基因对肾小管细胞HKC生长的影响。 结果 rAAV2-hNanog转染肾小管上皮细胞后，RT-PCR检测表明hNanog基因能在肾小管上皮细胞中稳定表达；同时，小管上皮细胞在转染后的增殖能力也显著增强（P < 0.05）。光镜下观察发现，转染的小管上皮细胞的形态无明显改变。 结论 利用hNanog基因提高生物人工肾小管种子细胞的增殖能力，可为生物人工肾的快速构建及应用提供一种新的方法。     

Long-term culture of glutamine synthetase-transfected HepG2 cells in circulatory flow bioreactor for development of a bioartificial liver

Glutamine synthetase (GS) is involved in an accessory pathway of ammonia removal in mammals. To develop a bioartificial liver with a human cell line, GS gene was transfected into HepG2 cells, which had no ammonia removal activity. After culturing in the presence of methionine sulfoximine (MSX), a GS inhibitor, we obtained a MSX-resistant HepG2 subline (GS-HepG2), which had amplified GS gene; ammonia removal activity was estimated to be 1/7 of that of rat primary culture hepatocytes. The cells were cultured in a circulatory flow bioreactor for 109 days, while they multiplied from 5 x 10(7) to 4 x 10(9) cells. Three days after inoculation, the ammonia level of the culture medium was lowered to a level maintained thereafter, suggesting that using recombinant cell lines for bioartificial livers enables long-term repeated treatment for hepatic failure patient. Judging from the rate of decrease in the amount of the added ammonia, the ammonia removal capability of 4 x 10(9) GS-HepG2 cells was almost equivalent to 5 x 10(8) porcine hepatocytes inoculated into the circulatory flow bioreactor. Apart from their ammonia removal activity, GS-HepG2 cells eliminated human tumor necrosis factor-alpha (TNF-alpha). Cytokine removal therefore promises to be another useful property of bioreactor cells.     

Hydrostatic pressure induces apoptosis of chondrocytes cultured in alginate beads.

The purpose of this study was to investigate the influence of hydrostatic pressure (HP) on apoptosis and expression of heat-shock protein 70 (HSP70) in chondrocytes cultured in alginate beads. Chondrocytes were isolated from the articular cartilage of rabbit joints and seeded in alginate beads. The beads in Group A were cultured for less than 24 h after being embedded with the chondrocytes, while those in Group B were cultured for 2 weeks. Both groups were exposed to HP of 10 or 50 MPa for 12 or 24 h. The beads in Groups A and B that were not exposed to HP were regarded as controls. Apoptotic cells induced by exposure to HP were quantified using the TUNEL method. Immunohistochemical analysis for HSP70 and in situ TUNEL analysis were also performed. Apoptotic chondrocytes were not observed in the control cells under atmospheric pressure, whereas apoptosis was observed in the beads in Group A, and the number of apoptotic cells increased as the duration and magnitude of HP increased. On the other hand, we observed no significant population of apoptotic cells in the beads in Group B. Chondrocytes expressing HSP70 were not TUNEL positive in the histological analysis. Excessively strong HP could evoke apoptosis when the extracellular matrix did not accumulate around the chondrocytes. HSP70 expression was related to occurrence of apoptosis that resulted from HP. These findings suggest a mechanism for the pathogenesis of cartilage degeneration in osteoarthritis.     

Effectiveness of synovial fluid mesenchymal stem cells embedded in alginate beads for treatment of steroid-induced avascular necrosis of the femoral head

Objective

Treatment of early stage avascular necrosis of the femoral head (AVNFH) is still challenging for clinicians today. Core decompression with the implantation of mesenchymal stem cells (MSCs) has become popular and been proven to be an effective therapy for ANNFH. Synovial fluid MSCs, which can be easily harvested by joint aspiration, reportedly have the potential to differentiate into bone and cartilage. The purpose of this investigation is to evaluate the effectiveness of core decompression plus the implantation of alginate beads embedded with synovial fluid MSCs (ABSMSCs) in bone regeneration in the treatment of steroid-induced AVNFH in a rabbit model.

Methods

An in vitro study is carried out to evaluate the bioactivity and osteogenic differentiation of synovial fluid MSCs in the environment formed by alginate beads. In an in vivo study, the application of ABSMSCs was combined with bone decompression to treat steroid-induced AVNFH in a rabbit model. Bone mineral density, radiography and histology were used to evaluate the bone growth of the femoral head after the rabbits had been euthanized 6 weeks after surgery.

Results

The results obtained in vitro showed that the synovial fluid MSCs in the environment of alginate beads had the potential to differentiate toward bone growth. In vivo, the treatment of steroid-induced AVNFH in a rabbit model by core decompression plus the implantation of ABSMSCs preserved the bone density and sphericity of the femoral head and promoted bone regeneration.

Conclusion

Implantation of ABSMSCs is a novel and effective therapy for AVNFH. Hopefully, this application will improve the outcome of early stage AVNFH and facilitate the harvesting of stem cells.     

In vivo study of biodegradable alginate antibiotic beads in rabbits.

The authors investigated the lyophilized poly-L-lysine-coated alginate antibiotic delivery system in vivo for the treatment of musculoskeletal infections. The sodium alginate was mixed with vancomycin, coated with poly-L-lysine and lyophilized to form 3 mm in diameter biodegradable antibiotic beads. The antibiotic beads were implanted in the distal femoral cavities of rabbits for in vivo investigation. The local concentration of vancomycin was well above the minimal inhibitory concentration of Staphylococcus aureus for 21 days. The release was most marked during the first two days. The diameters of sample inhibition zone ranged from 8 to 16 mm, the relative activity of vancomycin ranged from 12.5% to 100%. The blood level of vancomycin reached its peak (46.0 mg/l) two days after implantation and fell to 3.2 mg/l at two weeks. It was undetectable after three weeks. There was no increase in the concentration of blood urea nitrogen and serum creatinine after the implantation. Histological observations showed that bead materials were biodegradable, resorbed slowly and only cause mild host reaction. This study offers a biodegradable delivery system of antibiotics to treat musculoskeletal infections.     

In vivo estimation of bioartificial liver with recombinant HepG2 cells using pigs with ischemic liver failure

Biological efficacy of a recombinant human hepatic cell line, glutamine synthetase transfected HepG2 (GS-HepG2), was examined with large-scale culture in a circulatory flow bioreactor and in pigs with ischemic liver failure. GS-HepG2 cells were cultured in a circulatory flow bioreactor from 5 x 10(7) to 4 x 10(9) cells for 109 days. The cells showed ammonia removal activity even under substrate (glutamic acid)-free medium, suggesting that the GS catalyzed the activity using intracellular glutamic acid that had been pooled during conventional culture. When GS-HepG2 bioartificial liver (BAL) was applied to pigs with ischemic liver failure, survival time was prolonged to 18.8 +/- 6.1 h (mean +/- SD, n = 4) from 13.8 +/- 5.4 h (n = 6) and 10.7 +/- 4.1 h (n = 6) (groups treated with cell-free BAL and treated with plasma exchange and continuous hemodiafiltration, respectively). Laboratory data indicated the tendency for improvement in increase of blood ammonia level and decline of blood coagulation indices in the GS-HepG2 BAL-treated group. The advantages and potential for the cell line as a bioreactor in BAL is also discussed, comparing to those of isolated porcine hepatocytes.     

Radial flow bioreactor for the creation of bioartificial liver and kidney

A radial flow bioreactor (RFB) is used for a three-dimensional perfusion culture of hepatocellular carcinoma (HCC) cells and renal cells, to create a bioartificial liver and kidney. The cylindrical reactor is filled with porous cellulose microcarrier. RFB can be characterized as a system in which the medium flows from the periphery toward the center of the reactor, thereby delivering an adequate supply of oxygen and nutrients to cells at the center as well as at the periphery. HCC cells incubated in the RFB system at high density maintained viability for long periods of time. Proximal tubular cells (LLC-PK1) as well as HCC cells, but not human immortalized mesangial cells (HMC) were cultured in the RFB for more than 14 days. The mRNA expression of some enzymes involved in the urea cycle, cytochrome P450s in HCC cells, and the 1-alpha-hydroxylase (CYP27B1) in LLC-PK1 cells was higher than that in monolayer cultures. These results suggest that the RFB system composed of HCC cells or renal cells may be useful for a bioartificial liver and kidney.