형상이 제어된 골 조직 재생용 3차원 지지체를 적용한 새로운 형태의 Perfusion Culture

이시우 ( Shi Woo Lee ),허수진 ( Su Jin Heo ),장지연 ( Ji Yeon Jang ),정재영 ( Jae Young Jeong ),김수향 ( Su Hyang Kim ),박수아 ( Su A Park ),전은수 ( Eun Su Jeon ),신정욱 ( Jung Woog Shin ) 한국조직공학·재생의학회 2010 조직공학과 재생의학 Vol.7 No.1

The objective of this study was to investigate the efficacy of a newly designed perfusion bioreactor which provides flow-induced mechanical stimulation on the cells residing in the intra-morphology controllable scaffolds. For this, we fabricated scaffolds composed of poly e-caprolactone (PCL) and micro-sized hydroxyapatite (HA) particles using rapid-prototyping process. Also a new bioreactor system for perfusion culture was designed and developed. For the analyses of cellular responses of bone-tissue related cells cultured in the perfusion bioreactor system, various biological assays were performed such as MTT test, DNA content measurement, FE-SEM and live/dead staining. The cells used in this study were MG-63 (human osteoblast-like cell line) and mesenchymal stem cells from New Zealand white rabbits. Our results showed that the cells cultured by the perfusion bioreactor resulted in higher proliferation rate and mineralization of extracellular matrices than those cultured in static culture. From this study, we could confirm the potentials of 1) a newly developed perfusion bioreactor, 2) intra-morphology controllable scaffolds composed of PCL and HA particle and 3) the combination of the suggested scaffolds and perfusion culturing system in relation to bone tissue engineering.

Commercial-scale Economic Comparison of Different Batch Modes for Upstream and Downstream Processing of Monoclonal Antibody

차현명,Hwang Jeong-Min,연정흠,임진혁,한혜진,김동일 한국생물공학회 2021 Biotechnology and Bioprocess Engineering Vol.26 No.6

Chinese hamster ovary (CHO) cell cultures are widely used due to their high productivity, industrial track record, safety record, and correct post-translational modifications. Fed-batch and perfusion modes have been mostly adopted as traditional commercial manufacturing process using CHO cells for the production of biopharmaceuticals. Thus, we compared fed-batch and perfusion culture processes for the commercial-scale production of monoclonal antibody. Main production process was performed after confirming the comparable culture performance in seed train of different operation modes, and then perfusion cultures showed 1.6- fold higher peak viable cell density (VCD) with a longer culture duration than that of fed-batch cultures. Also, as the result of main batches, total product amount increased over 450% in perfusion cultures. Importantly, perfusion process allowed to improve the higher purification yield while maintaining acceptable product quality, because the level of process impurity could be reduced by using retention device. When economic comparison of fed-batch versus perfusion processes was carried out under the equal scale of upstream and downstream, perfusion mode was more a cost-effective bioprocess that could reduce the cost of goods. In conclusion, although comparison data of triplicate commercial batches are of course specific case for a given antibody production, this study verified the possibility of using perfusion mode as an efficient system for commercial producing the desired antibody and is a meaningful report in the biopharmaceutical industry.

액적의 균형공급에 의해 관류유량이 일정한 펌프 없는 세포배양 칩

김태윤(Taeyoon Kim),조영호(Young-Ho Cho) 대한기계학회 2011 大韓機械學會論文集B Vol.35 No.11

본 논문에서는 액적의 균형공급에 의해 관류유량이 일정하게 유지되는 펌프 없는 세포배양 칩을 제안하였다. 기존의 펌프 없는 세포배양 칩은 유체 수위차가 시간에 따라 점차 감소하여 일정한 관류유량 유지가 어려웠다. 반면, 제안된 칩은 액적의 균형공급으로 유체 수위차를 일정하게 유지하여 일정한 관류유량의 세포배양이 가능하다. 제작된 세포배양 칩의 성능분석 결과, 펌프 없이 최대 9.96%와 6.92%의 편차 및 오차 내에서 0.1~0.3μl/min 의 관류유량, Q, 을 얻었다. H358 폐암 세포주 배양결과, Q=0.2μl/min 의 관류유량에서 최대 57.8±21.1%/일의 증식률을 보여, Q=0μl/min 의 정치배양보다 1.9 배 높은 값을 얻었으며 활성도 또한 정치배양보다 관류배양이 더 높은 값을 보였다. 제안된 펌프 없는 세포배양 칩은 높은 증식률과 활성도의 좋은 배양환경을 제공하여 세포기반 바이오 분석에 응용 가능하다. We report on a pumpless cell culture chip in which a constant medium perfusion rate is maintained by balanced droplet dispensing. Previous chips had a decreasing perfusion rate due to the decreasing hydraulic-head difference ?h between the inlet and drain. However, the present chip maintains a constant medium perfusion rate due to the constant ?h between the inlet and drain maintained by balanced droplet dispensing. The perfusion rate Q was measured to be 0.1?0.3 μl/min with a maximum deviation and error of 9.96% and 6.92%, respectively. In the perfusion culture (Q = 0.1?0.3 μl/min), the maximum growth-rate of H358 cells was measured to be 57.8% ± 21.1% per day, which is 1.9 times higher than that of a static culture. The perfusion culture also resulted in higher cell viability than a static culture. The present chip offers a favorable environment with a high growth-rate and viability and thus has potential for use in the integrated cell culture system.

Two-Stage Depth Filter Perfusion Culture for Recombinant Antibody Production by Recombinant Chinese Hamster Ovary Cell

이준철,김도연,오덕재,장호남 한국생물공학회 2008 Biotechnology and Bioprocess Engineering Vol.13 No.5

A rCHO cell line of DUKX origin 26*-320, producing recombinant antibody against the human platelet, was cultivated in a two-stage depth filter perfusion system (DFPS) for 20 days in order to attain high recombinant antibody concentration. The productivity of the first stage DFPS bioreactor reached 53 times that of the batch culture in a controlled stirred tank reactor and was showed 12.1 mg/L antibody concentration at a perfusion rate of 6.0 d-¹. Glucose concentration in the first DFPS was maintained at 1.5 g/L to avoid cell damage in the perfusion culture. A second stage DFPS system was attached to the first DFPS, which resulted in a low glucose concentration of 0.02 g/L and a high antibody concentration of 23.9 mg/L. The two-stage depth filter perfusion culture yielded 60% higher product concentration than the batch and 49-fold higher produc-tivity of 69.3 mg/L/d in comparison with that (1.4 mg/L/d) in a batch system. Furthermore, antibody concentration of the sec-ond stage was 97% higher than that of the first stage, and the antibody productivities were comparable to that of the first stage. This two-stage DFPS system also showed potential for higher titer production of recombinant antibody and high volumetric productivity for long-term culture of bio-pharmaceutical substances.

Characteristics of human cell line, F2N78, for the production of recombinant antibody in fed-batch and perfusion cultures

Seo, J.S.,Min, B.S.,Kwon, Y.B.,Lee, S.Y.,Cho, J.M.,Park, K.H.,Yang, Y.J.,Maeng, K.E.,Chang, S.J.,Kim, D.I. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.121 No.3

<P>A human hybrid cell line, F2N78, was developed by somatic fusion of HEK293 and Namalwa cells for the production recombinant biopharmaceutical proteins. In this study, we performed perfusion culture to verify its potential in culture process used for human cell expression platform. Cell viability could be maintained over 90% and high viable cell density was obtained at higher than 1.0 x 10(7) cells/mL by bleeding process in perfusion culture. The cells were adapted well in both culture modes, but there were apparent differences in protein quality. Compared to fed-batch culture, degalactosylated forms such as G0F and G0 as well as Man5 showed no significant increases in perfusion culture. In terms of charge variants, acidic peaks increased, whereas main peaks constantly decreased according to the length of culture period in both methods. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.</P>

Modified harvest system for enhancing Factor VIII yield in alternating tangential flow perfusion culture

Kim, S.C.,An, S.,Kim, H.K.,Park, B.S.,Na, K.H.,Kim, B.G. Society for Bioscience and Bioengineering, Japan ; 2016 Journal of bioscience and bioengineering Vol.121 No.5

<P>This study describes the development and experimental verification of a modified harvest system to enhance Factor VIII (FVIII) yield in an alternating tangential flow (ATF) perfusion culture. The main innovation of the modified harvest system is the use of check and pinch valves, eliminating the need of a peristaltic pump for harvest. The system was applied to perfusion cultures of Chinese hamster ovary cells, which co-express both recombinant human FVIII (rhFVIII) and von Willebrand factor (vWF). The modified harvest system showed comparable cell growth with the conventional harvest system using a peristaltic pump. The perfusion rate was successfully controlled using the system. In addition, the modified harvest system achieved an approximately 13.6-fold increase in the final concentration yield of FVIII activity and a 1.47-fold increase in the production yield of FVIII activity compared with a peristaltic pump. Enhancement of the yield of FVIII activity resulted from the reduction of FVIII antigen (FVIII:Ag) retention. As a result of transmembrane pressure (TMP) measurement, the reduction of the retained FVIII:Ag was due to the increased TMP, which was caused by the characteristic function of a check valve, compared with a peristaltic harvest system. The modified harvest system developed in this study could be useful to enhance the production yield of other recombinant proteins in ATF perfusion culture. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.</P>

Bonding of Flexible Membranes for Perfusable Vascularized Networks Patch

Hong Soyoung,Song Yejin,최재순,황창모 한국조직공학과 재생의학회 2022 조직공학과 재생의학 Vol.19 No.2

BACKGROUND: In vitro generation of three-dimensional vessel network is crucial to investigate and possibly improve vascularization after implantation in vivo. This work has the purpose of engineering complex tissue regeneration of a vascular network including multiple cell-type, an extracellular matrix, and perfusability for clinical application. METHODS: The two electrospun membranes bonded with the vascular network shape are cultured with endothelial cells and medium flow through the engineered vascular network. The flexible membranes are bonded by amine-epoxy reaction and examined the perfusability with fluorescent beads. Also, the perfusion culture for 7 days of the endothelial cells is compared with static culture on the engineered vascular network membrane. RESULTS: The engineered membranes are showed perfusability through the vascular network, and the perfused network resulted in more cell proliferation and variation of the shear stress-related genes expression compared to the static culture. Also, for the generation of the complex vascularized network, pericytes are co-cultured with the engineered vascular network, which results in the Collagen I is expressed on the outer surface of the engineered structure. CONCLUSION: This study is showing the perfusable in vitro engineered vascular network with electrospun membrane. In further, the 3D vascularized network module can be expected as a platform for drug screening and regenerative medicine.

Dependence of Synchronized Bursting Activity on Medium Stirring and the Perfusion Rate in a Cultured Network of Neurons

Ryoun Heo,Hyun Kim,Kyoung J. Lee 한국물리학회 2016 THE JOURNAL OF THE KOREAN PHYSICAL SOCIETY Vol.68 No.9

A cultured network of neurons coupled with a multi-electrode-array (MEA) recording system has been a useful platform for investigating various issues in neuroscience and engineering. The neural activity supported by the system can be sensitive to environmental fluctuations, for example, in the medium’s nutrient composition, ph, and temperature, and to mechanical disturbances, yet this issue has not been the subject. Especially, a normal practice in maintaining neuronal cell cultures involves an intermittent sequence of medium exchanges, typically at a time interval of a few days, and one such sudden medium exchange is unavoidably accompanied by many unintended disturbances. Here, based on a quantitative time-series analysis of synchronized bursting events, we explicitly demonstrate that such a medium exchange can, indeed, bring a huge change in the existing neural activity. Subsequently, we develop a medium perfusion-stirring system and an ideal protocol that can be used in conjunction with a MEA recording system, providing long-term stability. Specifically, we systematically evaluate the effects of medium stirring and perfusion rates. Unexpectedly, even some vigorous mechanical agitations do not have any impacts on neural activity. On the other hand, too much replenishment (e.g., 1.8 ml/day for a 1.8-ml dish) of neurobasal medium results in an excitotoxicity.

Mathematical analysis of colonial formation of embryonic stem cells in microfluidic system

민슬기,신화성,Byung Man Lee,Jin Ha Hwang,하성호 한국화학공학회 2012 Korean Journal of Chemical Engineering Vol.29 No.3

A fluidic environment affects mechanochemical characteristics of embryonic stem cells (ESCs). Perfusion is recognized as an attractive culture mode of ESCs since the steady fluidic state can enhance ESCs’ controllability,supporting a unique cell culture condition. Cellular membrane motility presents important information about cellular dynamics such as adhesion, spreading, and migration. Thus, an investigation of the perfusion-induced membrane motility is significant to understand the mechanochemical behavior of ESCs in the steady culture state. In this research,we suggest Lfr, the ratio of circumferential membrane unattached to other cells’ to the cell’s circumference, as a new parameter to characterize cells’ shape and motility. Lfr of embryonic stem cells has positive correlations with cellular area (Ar) and free peripheral length (Lf) but a negative correlation with roundness (Rn). We also propose a mathematical model representing ESCs’ membrane motilities and demonstrate their colonical behavior.

타액선 도관세포의 관류 배양 기술 개발

김지원,김정미,최정석 대한갑상선학회 2018 International Journal of Thyroidology Vol.11 No.2

Background and objectives: Salivary hypofunction is one of the common side effects after radioiodine therapy, and its pathophysiology is salivary ductal stenosis resulting from ductal cell injury. This study aimed to develop the functional culture environment of human parotid gland ductal cells in in vitro three-dimensional perfusion culture system. Materials and Methods: We compared plastic dish culture method and three-dimensional culture system containing Matrigel and nanofiber. Morphogenesis of reconstituted salivary structures was assessed by histomorphometry. Functional characteristics were assessed by immunohistochemistry and reverse transcription polymerase chain reaction (aquaporin 5, CK7, CK18, connexin 43, and p21). In addition, we designed the media perfusion culture system and identified higher rate of cell proliferation and expression of connexin 43 in perfusion system comparing to dish. Results: Human parotid ductal cells were well proliferated with the ductal cell characters under environment with Matrigel. In the presence of Matrigel, aquaporin 5, CK18 and connexin 43 were more expressed than 2D dish and 3D nanofiber setting. In the media perfusion culture system, ductal cells in 3D culture media showed higher cells count and connexin 43 expression compared to 2D dish. Conclusion: This in vitro ductal cell perfusion culture system using Matrigel could be used to study for radioiodine induced sialadenitis model in vivo.