• Title/Summary/Keyword: perfusion culture

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Effect of Fish Serum on the Primary Monolayer Culture of Catfish (Silurus asotus) Hepatocytes (어류혈청이 메기(Silurus asotus) 간세포의 단층배양에 미치는 영향)

  • Kwon, Hyuk-Chu;Choi, Seong-Hee;Kim, Eun-Hee;Han, Deug-Woo;Kwon, Joon-Yeong
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.39 no.1
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    • pp.23-26
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    • 2006
  • Effects of sera from several fish species and insulin on the development of cultured Silurus asotus hepatocytes were investigated. Hepatocytes with high viability (95%) were obtained from the livers of male catfish by two step collagenase perfusion. Isolated hepatocytes, initially showed a typical round-shape, firmly attached to the culture dish within 24 h. In the presence of catfish serum, hepatocytes attached each other, spread well on the dish and developed into monolayer after 3-4 days of incubation. Cells within the established monolayer became polygonal in shape and their nuclei and boundaries being clearly visible under the microscope. In contrast, when incubated in FBS-supplemented or serum-free medium, cells managed to form small clusters, each made of 2-10 cells. Cells in FBS-supplemented medium further developed into larger clusters. However, these clusters failed to develope into monolayer. In addition, when insulin was deprived from culture medium, formation of monolayer also failed. From these data, it can be concluded that the presence of both catfish serum and insulin is necessary for the formation of monolayer of catfish hepatocytes and the functional role of fish serum may differ from that of insulin and can not be displaced by FBS-supplementation.

Glucosamine Hydrochloride and N-Acetylglucosamine Influence the Response of Bovine Chondrocytes to TGF-β3 and IGF in Monolayer and Three-Dimensional Tissue Culture

  • Pizzolatti, Andre Luiz A.;Gaudig, Florian;Seitz, Daniel;Roesler, Carlos R.M.;Salmoria, Gean Vitor
    • Tissue Engineering and Regenerative Medicine
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    • v.15 no.6
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    • pp.781-791
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    • 2018
  • BACKGROUND: Glucosamine hydrochloride (GlcN HCl) has been shown to inhibit cell growth and matrix synthesis, but not with N-acetyl-glucosamine (GlcNAc) supplementation. This effect might be related to an inhibition of critical growth factors (GF), or to a different metabolization of the two glucosamine derivatives. The aim of the present study was to evaluate the synergy between GlcN HCl, GlcNAc, and GF on proliferation and cartilage matrix synthesis. METHOD: Bovine chondrocytes were cultivated in monolayers for 48 h and in three-dimensional (3D) chitosan scaffolds for 30 days in perfusion bioreactors. Serum-free (SF) medium was supplemented with either growth factors (GF) $TGF-{\beta}$ ($5ng\;mL^{-1}$) and IGF-I ($10ng\;mL^{-1}$), GlcN HCl or GlcNAc at 1mM each or both. Six groups were compared according to medium supplementation: (a) SF control; (b) SF + GlcN HCl; (c) SF + GlcNAc; (d) SF + GF; (e) SF + GF + GlcN HCl; and (f) SF + GF + GlcNAc. Cell proliferation, proteoglycan, collagen I (COL1), and collagen II (COL2) synthesis were evaluated. RESULTS: The two glucosamines showed opposite effects in monolayer culture: GlcN HCl significantly reduced proliferation and GlcNAc significantly augmented cellular metabolism. In the 30 days 3D culture, the GlcN HCl added to GF stimulated cell proliferation more than when compared to GF only, but the proteoglycan synthesis was smaller than GF. However, GlcNAc added to GF improved the cell proliferation and proteoglycan synthesis more than when compared to GF and GF/GlcN HCl. The synthesis of COL1 and COL2 was observed in all groups containing GF. CONCLUSION: GlcN HCl and GlcNAc increased cell growth and stimulated COL2 synthesis in long-time 3D culture. However, only GlcNAc added to GF improved proteoglycan synthesis.

Optimization and Mathematical Modeling of the Transtubular Bioreactor for the Production of Monoclonal Antibodies from a Hybridoma Cell Line

  • Halberstadt, Craig R.;Palsson, Bernhanrd O.;Midgley, A.Rees;Curl, Rane L.
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.7 no.3
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    • pp.163-170
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    • 2002
  • This report describes the use of a transtubular bioreactor to study the relative effects of diffusion versus perfusion of medium on antibody production by a hybridoma cell line. The study was performed with a high-density cell culture maintained in a serum-free, low-protein medium for 77 days. It was determined that the reactor possessed a macro-mixing pattern residence time distribution similar to a continuous stirred tank reactor (CSTR), However, due to the arrangement of the medium lines in the reactor, the flow patterns for nutrient distribution consist of largely independent medium path lengths ranging from short to long. When operated with cyclic, reversing, transtubular medium flow, some regions of the reactor (with short residence times) are more accessible to medium than others (with long residence times). From this standpoint, the reactor can be divided into three regions: a captive volume, which consists of medium primarily delivered via diffusion; a lapped volume, which provides nutrients through unilateral convection; and a swept volume, which operates through bilateral convection. The relative sizes of these three volumes were modified experimentally by changing the period over which the direction of medium flow was reversed from 15 min (larger captive volume) to 9 h (larger swept volume). The results suggest that antibody concentration increases as the size of the diffusion-limited (captive) volume is increased to a maximum at around 30 min with a sharp decrease thereafter. As reflected by changes in measured consumption of glucose and production of lactate, no significant difference in cellular metabolism occurred as the reactor was moved between these different states. These results indicate that the mode of operation of the transtubular bioreactor may influence antibody productivity under serum-free, low-protein conditions with minimal effects on cellular metabolism.

Use of Moving Aeration Membrane Bioreactor for the Efficient Production of Tissue Type Plasminogen Activator in Serum Free Medium

  • Hyun Koo Kim;Moo
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.1 no.1
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    • pp.32-35
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    • 1996
  • Amoving aeration-membrane (MAM) bioreactor was employed for the production of 2$\mu$g/mL of tissue type Plasminogen Activator (tPA)in serum free medium from normal human fibroblast cells. This system could maintain high cell density for long periods of steady state conditions in perfusion cultivation. Under normal operating condition, shear stress was as low as 0.65 dynes/$\textrm{cm}^2$ at the agitation speed of 80 rpm. Even though cell density gradually decreased with increasing agitation speed, tPA production increased linearly with increasing shear stress within a moderate range. This culture system allowed production of 2$\mu$g tPA/mL while maintaining a high cell denisty of 1.0$\times$107 viable cell/mL.

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Isolated Native Valve Endocarditis on Tricuspid Valve -A case report- (자기 삼첨판막에 발생한 고립성 심내막염의 외과적 치료 -1예보고-)

  • 홍준화;소동문;정조원;홍창호
    • Journal of Chest Surgery
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    • v.32 no.12
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    • pp.1119-1122
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    • 1999
  • Native valve endocarditis (NVE) without preexisting structural valve or congenital cardiac malformation especially in pediatric group is rare. A case of isolated tricuspid valve endocarditis in a 7-year-old child without any cardiac malformation is described. This child had suffered from fever and productive cough for 3 weeks. Blood culture grew Staphylococcus aureus. Fever was not controlled even with proper antibiotic treatment. Transthoracic echocardiogram and lung perfusion scan revealed a large vegetation on the tricuspid valve with multiple embolism Surgical procedures included vegetectomy partial cusps resection and pericardial patch valvuloplasty. Th patient was in NYHA class I during follow up.

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Effect of Ginseng Components (Ginsenosides and Fat Soluble Fraction) on Rat Liver Glucokinase Activity (쥐의 간 Glucokinase 활성에 미치는 인삼 성분의 영향)

  • 주충노;김선진
    • Journal of Ginseng Research
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    • v.18 no.1
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    • pp.17-24
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    • 1994
  • Effect of ginsenoside mixture, ginsenoside $Rb_1$,$Rb_2$,$Rg_1$ and the fat soluble fraction of the roots of Panax ginseng C.A. Meyer on the activity of glucokinase (GK) in vitro has been observed and found that GK activity was increased about 15c1c at the concentration of ginsenoside mixture and/or the fat soluble fraction being $10^{-7}$,$10^{-5}$%. It was also observed that glucose uptake by rat liver was increased in the presence of either ginsenoside mixture or the fat soluble fraction by perfusion technique. Ginsenoside mixture stimulated various enzymes related to glucose metabolism, however, both ginsenoside mixture and the fat soluble fraction did not stimulate GK activity as expected. Primary culture of liver cells showed that the ginsenoside mixture and the fat soluble fraction increased GK activity significantly and they stimulated the GK activity synergistically in the co-presence of insulin.

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Economic Production of $\gamma$-Interferon from Recombinant Human Cells in Serum Free Medium by a Moving Aeration Membrane Bioreactor (교반형 막 반응기를 이용한 재조합 인간 세포의 무혈청 배지에 의한 $\gamma$-Interferon의 생산)

  • Park, Young-Shik;Kim, Hyun-Kyu;Lim, Seo-Kyu;Park, Kyung-You;Lee, Hyeon-Yong
    • Microbiology and Biotechnology Letters
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    • v.22 no.4
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    • pp.389-394
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    • 1994
  • 8 X 10$^{6}$(viable cells/ml) of maximum cell density and 9000(IU/ml) of $\gamma$-IFN production were obtained at 55(ml/hr) of a perfusion rate by cultivating HSF cells using a moving membrane aeration bioreactor. This system proves to be an efficient culture process by maintaning 90% of viable cells during the whole cultivation periods. The metabolic molar quotient of glucose to lactate was 0.81 for overall ranges of glucose consumed while the evolution of ammonia was not linearly related to the consumption of glutamine. Low molar conversion ratio was observed in low consumptions of glutamine and high molar conversion ratio in high comsumptions. It also shows that the glutamolysis plays important role in the steady state conditions by evolving larger quantities of ammonia than lactate. At the above of 50 rpm, which is the optimum agitation speed for this bioreactor, the cell growth was severely affected while the IFN production was less decrea- sed, maintaing 1.5 X 10$^{-3}$(IU/cell/day) specific IFN production rate. The cumulatvie $\gamma$-IFN production was 7.2 X 10$^{8}$(IU) for 70 days of the cultivation, which yields 1 X 10$^{7}$ (IU/day) of IFN production rate. Therefore, a commercial production of $\gamma$-IFN by this culture process can be achievable by maintaining the above IFN productivity in a scaled-up culture system.

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Inhibitory effects of KHG26377 on glutamate dehydrogenase activity in cultured islets

  • Yang, Seung-Ju;Hahn, Hoh-Gyu;Choi, Soo-Young;Cho, Sung-Woo
    • BMB Reports
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    • v.43 no.4
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    • pp.245-249
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    • 2010
  • GDH has been known to be related with hyperinsulinism-hyperammonemia syndrome. We have screened new drugs with a view to developing effective drugs modulating GDH activity. In the present work, we investigated the effects of a new drug, KHG26377 on glutamate formation and GDH activity in cultured rat islets. When KHG26377 was added to the culture medium for 24 h prior to kinetic analysis, the $V_{max}$ of GDH was decreased by 59% whereas $K_m$ is not significantly changed. The concentration of glutamate decreased by 50% and perfusion of islets with KHG26377 reduced insulin release by up to 55%. Our results show that KHG26377 regulates insulin release by inhibiting GDH activity in primary cultured islets and support the previous studies for the connection between GDH activity and insulin release. Further studies are required to determine in vivo effects and pharmacokinetics of the drug.

The Adjunctive Role of Resectional Surgery for the Treatment of Multidrug-Resistant Pulmonary Tuberculosis (다제내성 폐결핵의 치료에서 폐절제술의 보조적인 역할)

  • Koh, Won-Jung;Lee, Jae-Ho;Yoo, Chul-Gyu;Kim, Young-Whan;Chung, Hee-Soon;Sung, Sook-Whan;Im, Jung-Gi;Kim, Joo-Hyun;Shim, Young-Soo;Han, Sung-Koo
    • Tuberculosis and Respiratory Diseases
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    • v.44 no.5
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    • pp.975-991
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    • 1997
  • Background : Many patients with isoniazid and rifampin-resistant pulmonary tuberculosis have organisms that are also resistant to other first-line drugs. Despite of aggressive retreatment chemotherapy, the results are often unsuccessful, with a failure rate approaching 40%. Recently, there has been a revival of resectional surgery for the treatment of multidrug-resistant pulmonary tuberculosis. Methods : A retrospective analyses of the case records and radiographic findings were done. Between January 1991 and December 1995, 14 human immunodeficiency virus (HIV)-seronegative patients with multidrug-resistant pulmonary tuberculosis were selected for resection to supplement chemotherapy. All patients had organisms resistant to many of the first-line drugs, including both isoniazid and rifampin. Results : Despite of aggressive therapy for median duration of 9.5 months, 12 of the 14 patients (86%) were still sputum smear and/or culture positive at the time of surgery. The disease was generally extensive. Although main lesions of the disease including thick-walled cavities were localized in one lung, lesser amounts of contralateral disease were demonstrated in 10 of 14 (71%). Types of surgery performed were pneumonectomy including extrapleural pneumonectomy in six patients, lobectomy or lobectomy plus in six patients, and segmentectomy in two patients. The resected lung appeared to have poor function ; preoperative perfusion lung scan showed only 4.8% of the total perfusion to the resected portion of the lung. There were no operative deaths. Two patients had major postoperative complications including empyema with bronchopleural fistula and prolonged air leak, respectively. Of the 14 patients, 13 (93%) remained sputum-culture-negative for M. tuberculosis for a median duration of 23 months and one remained continuously sputum smear and culture positive for M. tuberculosis. Conclusion : On the basis of comparison with historical controls, adjunctive resectional surgery appears to play a significant beneficial role in the management of patients with multidrug-resistant pulmonary tuberculosis if the disease is localized and there are adequate reserve in pulmonary function.

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재조합 단백질 생산을 위한 식물세포 고농도 배양과 기체조성에 따른 영향

  • Lee, Sang-Yun;Jeon, Su-Hwan;Min, Byeong-Hyeok;Heo, Won;Jo, Gyu-Heon;Kim, Dong-Il
    • 한국생물공학회:학술대회논문집
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    • 2000.11a
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    • pp.425-428
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    • 2000
  • To enhance the productivity of recombinant protein, ${\beta} -glucuronidase(GUS)$, by transgenic Nicotiana suspensions, perfusion culture was carried out in a 5-L stirred tank bioreactor. Maximum cell density of 20.0 gDCW/L was obtained and GUS activity was noticeably affected by medium composition, such as salt concentration. Effect of headspace purging by $CO_2-enriched$ air was also investigated. Aeration of $CO_2(5%)-enriched$ air with 0.2 vvm significantly promoted the cell growth in lag pahse and increased volumetric GUS activity from 0.69 U/mL to 5.76 U/mL. In contrast, aeration of $CO_2(5%)-enriched$ air with 0.1 vvm did not affect the cell growth but enhanced the GUS acitivity from 3.24 to 5.25 U/mL at the 4th day.

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