온도감응성 마이크로캐리어를 이용한 세포 계대배양

양희석,김병수,서지혜 한국조직공학과 재생의학회 2009 조직공학과 재생의학 Vol.6 No.13

Microcarrier culture systems are frequently used for large-scale culture of anchorage-dependent mammalian cells. Mass production of a large amount of cells needs serial propagation in microcarrier culture. This requires enzyme treatment which could be a complex step in large-scale cell culture and cause cell damage. Thus, we have developed thermo-sensitive microcarriers by incorporating poly-N-isopropylacrylamide (pNIPAAm) onto Cytodex-3® (pNIPAAm-Cytodex). In this study, we tested the feasibility of serial propagation of Vero cells cultured on pNIPAAm-Cytodex microcarriers in three-dimensional bioreactors. Cells cultured on pNIPAAm-Cytodex were detached from pNIPAAm-Cytodex by a moderate change in temperature without enzyme treatment. This would simplify the complex serial propagation steps, avoid cell damage possibly caused by enzyme treatment, and enable one to easily harvest cells, because cells can be detached from microcarriers without enzyme treatment. Vero cells adhered, spread, and grew successfully on the thermo-sensitive microcarriers similarly to Cytodex microcarriers, and were harvested by simple temperature changes, thereby avoiding the use of proteolytic enzymes and laborious and time-consuming processes. Moreover, Vero cells detached by temperature change reattached and grew on pNIPAAm-Cytodex after scale-up in a manner similar to culture on Cytodex. The pNIPAAm-Cytodex microcarrier culture method would be useful for the mass production of biologicals through large-scale suspension culture of anchorage-dependent mammalian cells. Microcarrier culture systems are frequently used for large-scale culture of anchorage-dependent mammalian cells. Mass production of a large amount of cells needs serial propagation in microcarrier culture. This requires enzyme treatment which could be a complex step in large-scale cell culture and cause cell damage. Thus, we have developed thermo-sensitive microcarriers by incorporating poly-N-isopropylacrylamide (pNIPAAm) onto Cytodex-3® (pNIPAAm-Cytodex). In this study, we tested the feasibility of serial propagation of Vero cells cultured on pNIPAAm-Cytodex microcarriers in three-dimensional bioreactors. Cells cultured on pNIPAAm-Cytodex were detached from pNIPAAm-Cytodex by a moderate change in temperature without enzyme treatment. This would simplify the complex serial propagation steps, avoid cell damage possibly caused by enzyme treatment, and enable one to easily harvest cells, because cells can be detached from microcarriers without enzyme treatment. Vero cells adhered, spread, and grew successfully on the thermo-sensitive microcarriers similarly to Cytodex microcarriers, and were harvested by simple temperature changes, thereby avoiding the use of proteolytic enzymes and laborious and time-consuming processes. Moreover, Vero cells detached by temperature change reattached and grew on pNIPAAm-Cytodex after scale-up in a manner similar to culture on Cytodex. The pNIPAAm-Cytodex microcarrier culture method would be useful for the mass production of biologicals through large-scale suspension culture of anchorage-dependent mammalian cells.

Effects of Feeder Cells on the Primary Culture of Ovarian Cell Populations from Adult Japanese Medaka (Oryzias latipes)

Jun Hyung Ryu,Seung Pyo Gong 한국동물생명공학회(구 한국동물번식학회) 2020 Journal of Animal Reproduction and Biotechnology Vol.35 No.1

Fish ovarian germline stem cells (OGSCs) that have the abilities to self-renew and differentiate into functional gametes can be used in various researches and applications. A main issue to be solved for effective utilization of fish OGSCs is the development of their stable in vitro culture condition, but only few researches about fish OGSC culture have been reported so far. In this study, in order to find the clues to develop the culture condition for OGSCs from Japanese medaka (Oryzias latipes), we tried to establish somatic cell lines as a candidate for the feeder cells and evaluated its supporting effects on the culture of ovarian cell populations from O. latipes. As the results, the somatic cell lines could be established only from the embryonic tissues among three tissues derived from embryos, fins and ovaries. Three embryonic cell lines were tested as a feeder cell for the culture of ovarian cell population and all three cell lines induced cell aggregation formation of the cultured ovarian cells whereas the feeder-free condition did not. Furthermore, a significant cellular proliferation was observed in the ovarian cells cultured on two of three cell lines. As a trial to increase the capacity of the cell lines as a feeder cell that supports the proliferation of the cultured ovarian cells, we subsequently established a stable line that expresses the foreign O. latipes fibroblast growth factor 2 (FGF2) from an embryonic cell line and evaluated its effectiveness as a feeder cell. The ovarian cells cultured on FGF2 expressing feeder cells still formed cell aggregates but did not show a significant increase in cellular proliferation compared to those cultured on non-transformed feeder cells. The results from this study will provide the fundamental information for in vitro culture of medaka OGSCs.

Suspension culture of Vero cells for the production of adenovirus type 5

이득기,박지혜,서동원 대한백신학회 2020 Clinical and Experimental Vaccine Research Vol.9 No.1

Purpose: Most cell culture processes for viral vaccine production are mainly based on adherent cell culture systems using serum, which are associated with expensive and labor-intensive processes to produce large amounts of viral vaccine strains. In this study, we investigated whether Vero cells could be grown in serum-free and shaking suspension conditions. Furthermore, we assessed the ability of the Vero cell suspension culture system to produce adenovirus type 5 (Ad5), compared to that of the adhesive Vero cell culture system. Materials and Methods: We tested the feasibility of commercial serum-free media for Vero cell culture. For the adaptation of Vero cells in suspension culture, adhesive Vero cells were added in the early phase of shaking suspension culture, and 50 days after shaking suspension culture, suspension-adapted Vero cells were subcultured continuously. To assess the virus production ability of Vero cells in suspension, the cells were infected with Ad5-green fluorescent protein and evaluated based on their fluorescence intensity. Results: The Vero cells grown in OptiPRO serum-free medium showed no changes in morphology and growth rate, but MRC-5 and FRhk-4 cells showed morphological changes and decreased growth rate, respectively. The Vero cells were well adapted to the suspension culture system. The Vero cells in suspension showed a better Ad5 production ability than the adherent Vero cells. Conclusion: Vero cells can be grown in OptiPRO serum-free medium. Further, our suspension culture-adapted Vero cells may be suitable to produce viral vaccine strains due to their high ability to produce viruses such as Ad5.

Paper-based Cell Culture Microfluidic System

Fang Fang Tao,Xia Xiao,Kin Fong Lei,I-Chi Lee 한국바이오칩학회 2015 BioChip Journal Vol.9 No.2

In the past decades, glass/PDMS-basedmicrofluidic systems have been rapidly developed to provide homogenous and stable microenvironment for culturing cells. Although these excellent demonstrations involve much simplified operations than traditional cell culture protocol, but they are still not readily accessible to untrained personnel and not appropriate to operate in conventional biological laboratories. In this work, cellulose filter papers were used for the substrates of the cell culture microfluidic system, which provides a convenient tool for cell-based assay. A paper was patterned with culture areas and channels by wax printing technique. Medium or tested substance can be passively perfused to the culture areas. Analyses of cyto-compatibility, cell proliferation, cell morphology, and cell chemosensitivity were performed to confirm the possibility of the paper-based system. Theculture system could provide a platform for a wide range of cell-based assays with applications in drug screening and quantitative cell biology. This work demonstrated a paper-based cell culture microfluidic system and the system is inexpensive, disposable, and compatible to the existing culture facility. In the past decades, glass/PDMS-based microfluidic systems have been rapidly developed to provide homogenous and stable microenvironment for culturing cells. Although these excellent demonstrations involve much simplified operations than traditional cell culture protocol, but they are still not readily accessible to untrained personnel and not appropriate to operate in conventional biological laboratories. In this work, cellulose filter papers were used for the substrates of the cell culture microfluidic system, which provides a convenient tool for cell-based assay. A paper was patterned with culture areas and channels by wax printing technique. Medium or tested substance can be passively perfused to the culture areas. Analyses of cyto-compatibility, cell proliferation, cell morphology,and cell chemosensitivity were performed to confirm the possibility of the paper-based system. The culture system could provide a platform for a wide range of cell-based assays with applications in drug screening and quantitative cell biology. This work demonstrateda paper-based cell culture microfluidic system and the system is inexpensive, disposable, and compatible to the existing culture facility.

Fluidized Bed Culture System Using Microcarriers for Adherent Cells

Seohyun PARK,Jinwoo LEE,Duk Jae OH 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Adherent cells are the most common type of animal cells. They are used in many fields such as cell therapy, regenerative medicine, and the production of biological products. Thus the demand for them is also growing significantly. But the typical stationery (two-dimensional, 2-D) cell culture system is not suitable for large-scale culturing of anchorage-dependent cells due to a limited surface area for cell attachment and lack of control for the culture environments. To overcome these issues, we used microcarriers with a high surface-to-volume. Most cell cultures using microcarriers are cultivated in bioreactor systems. However, the impeller of the bioreactor required to maintain homogenous culture conditions causes turbulent flows and high shear forces, which induce negative effects on the cells being cultured. Therefore, we tried to cultivate adherent cells using a non-impeller type 3-D culture system with liquid flows for floating and mixing of beads, fluidized bed. In the fluidized bed cell culture system, the cells well adhered to the microcarriers until the end of the culture, and the cells grew well. The fluidized bed cell culture system can be simply scaled up just by increasing the volume or size of the column so it can be an option for mass culture of adherent cells.

지주세포증후군 환자에서 생식세포 배양의 유용성

오종진,홍재엽,임정진,이동률,홍영권 대한비뇨의학회 2009 Investigative and Clinical Urology Vol.50 No.3

Purpose: We determined the usefulness of in vitro germ cell culture in nonobstructive azoospermic patients diagnosed with Sertoli cell only syndrome, no sperm in testicular sperm extraction. Materials and Methods: This study included 44 patients (45 testicular tissues) with nonobstructive azoospermia who were diagnosed with Sertoli cell only syndrome and were found to have no sperm in testicular sperm extraction between January 2006 and July 2008. Among the 45 testicular tissues, 22 tissues were processed for culture. In the in vitro cultures, the testicular tissues were dissociated and plated on gelatin-coated dishes. Patients were divided into 2 groups according to culture success: group I, culture positive (+; n=10); and group II, culture negative (−; n=12). Results: The mean patient ages were 31.73 and 31.68 years for groups I and II, respectively. The mean testicular sizes were 10.19 and 10.42 cc, respectively; the semen volumes were 2.86 and 3.04 cc, respectively; and the mean FSH, LH, and testosterone levels were 18.86 mIU/ml, 5.99 mIU/ml, and 4.46 ng/ml vs. 21.02 mIU/ml, 6.29 mIU/ml, and 4.32 ng/ml for groups I and II, respectively, with no significant differences between the groups (p>0.05). The culture rate of nonobstructive azoospermic patients diagnosed with Sertoli cell only syndrome was 45.5% (10/22). Round spermatid injection was done in 2 patients with consent of the patients, but implantation failed. Among the 45 tissues, germ cells were found in 8 tissues after pathologic reexamination. Conclusions: The in vitro culture of germ cells would be useful in the advanced treatment of nonobstructive azoospermic patients. Purpose: We determined the usefulness of in vitro germ cell culture in nonobstructive azoospermic patients diagnosed with Sertoli cell only syndrome, no sperm in testicular sperm extraction. Materials and Methods: This study included 44 patients (45 testicular tissues) with nonobstructive azoospermia who were diagnosed with Sertoli cell only syndrome and were found to have no sperm in testicular sperm extraction between January 2006 and July 2008. Among the 45 testicular tissues, 22 tissues were processed for culture. In the in vitro cultures, the testicular tissues were dissociated and plated on gelatin-coated dishes. Patients were divided into 2 groups according to culture success: group I, culture positive (+; n=10); and group II, culture negative (−; n=12). Results: The mean patient ages were 31.73 and 31.68 years for groups I and II, respectively. The mean testicular sizes were 10.19 and 10.42 cc, respectively; the semen volumes were 2.86 and 3.04 cc, respectively; and the mean FSH, LH, and testosterone levels were 18.86 mIU/ml, 5.99 mIU/ml, and 4.46 ng/ml vs. 21.02 mIU/ml, 6.29 mIU/ml, and 4.32 ng/ml for groups I and II, respectively, with no significant differences between the groups (p>0.05). The culture rate of nonobstructive azoospermic patients diagnosed with Sertoli cell only syndrome was 45.5% (10/22). Round spermatid injection was done in 2 patients with consent of the patients, but implantation failed. Among the 45 tissues, germ cells were found in 8 tissues after pathologic reexamination. Conclusions: The in vitro culture of germ cells would be useful in the advanced treatment of nonobstructive azoospermic patients.

골수세포의 단기배양을 통한 조혈전구세포의 증폭

오덕연,김현수,박상준,김종숙,최지영,윤한중,김삼용 大韓血液學會 1996 大韓血液學會誌 Vol.31 No.2

성장생물학 : 지방과 근육 세포주의 단독 및 공동배양을 통한 세포형태학 및 세포물질 비교 연구

연성흠 ( Seong Heum Yeon ),조원모 ( Won Mo Cho ),백경훈 ( Kyung Hoon Baek ),송만강 ( Man Kang Song ),최창원 ( Chang Weon Choi ),황보순 ( Bo Soon Hwang ),박성권 ( Sung Kwon Park ) 한국동물자원과학회 ( 구 한국축산학회 ) 2012 한국축산학회지 Vol.54 No.2

본 연구는 기존 단독배양 위주로 이루어져온 세포배양 연구의 방법학적 한계의 극복과 대안을 제시하고자 지방과 근육세포주의 단독 및 공동배양에서 배양기법에 따른 지방 및 근육세포의 분화에 미치는 영향을 비교 조사하고자 실시하였다. 3T3-L1(지방세포) 및 L6(근육세포) 세포주는 성장배지인 10% FBS/DMEM(1% Pen- Strep solution 및 0.1% Fungizone 첨가) 하에서 48h 동안 단독배양 후 5% FBS/DMEM에서 배양하였다. 분화를 위한 단독 및 공동배양에서는 지방 및 근육세포 모두 분화유도물질 없이 2% FBS/DMEM으로 배양하였고, 공동배양에서는 0.4㎛ insert membrane을 사용하여 6 well plate 하단에 L6 cell을, 상단에는 3T3-L1 cell을 공생시켰다. 지방 및 근육세포 분화정도 측정은 세포별 형태학적 측정과 glycerol-3-phosphate dehydrogenase (GPDH) 및 creatine kinase(CK) 분석을 통해 조사되었다. 형태학적으로 볼때 3T3-L1 세포주는 공동배양보다 단독배양 시 분화가 더욱 잘 일어났고 L6 세포주의 경우 역으로 같았다. 세포물질분석에서는 분화배지 처리일(day 0)과 비교해 단독 및 공동배양 모두 지방세포 내 GPDH의 활성도가 유의적으로(P<0.05) 증가했음을 확인할 수 있었고 단독배양이 공동배양보다 유의적으로(P<0.05) 높은 수준의 GPDH 활성도를 보였다. L6 역시 마찬가지로 분화배지 처리일에 비하여 단독 및 공동배양 모두 CK 활성도가 유의적으로(P<0.05) 높았고, CK 활성도가 공동배양에서 유의적으로(P<0.05) 높게 나타남을 확인할 수 있었다. 이러한 결과는 기존 연구에서 이용된 단독 배양을 통한 세포 분화 결과 등은 생체와 비교 시 방법학적 한계로 인해 실제 생체 내에서는 그 분화정도가 매우 다를 것으로 생각되며, 이것은 앞으로 정확한 세포배양 결과 확보를 위해서는 단독배양보다는 공동배양기법을 사용해야 함을 의미한다. 향후 다양한 조건과 분화조절 물질들의 첨가를 통한 추가적인 공동배양실험이나 지방분화관련 분자생물학적 물질분석 등 다양한 실험 수행 시 보다 현실적이고 대량의 기초자료 확보가 가능할 것으로 판단된다. Present study was performed to investigate the effect of single and co-culture of adipocyte and muscle cell lines on cell differentiation. 3T3-L1(adipocyte) and L6(muscle) cell lines were single-cultured on the condition of 10% fetal bovine serum (FBS)/Dulbeco`s modified eagle`s medium(DMEM) for 48 h followed by culture within 5% FBS/DMEM as a growth media. Then, the growth media was replaced by differentiation media composed of 2% FBS/DMEM without additives in single- or co-culture of the 3T3-L1 and the L6 cells to induce differentiation of both cell types. In co-culture system, the 3T3-L1 and the L6 cells were grown in separated places by being seeded on a 0.4μm insert membrane and on the bottom of 6 well plate, respectively. Cell differentiation was measured using morphological investigation and cytosolic analysis of glycerol-3-phosphate dehydrogenase(GPDH; for 3T3-L1) and creatine kinase(CK; for L6). Based on the GPDH results, the presence of L6 cells did not stimulate 3T3-L1 differentiation showing more differentiation of 3T3-L1 cells in the single-culture compared to the co-culture condition. In contrast, 3T3-L1 cells in the co-culture promoted differentiation of L6 cells. Enzymatic analysis supported this result showing that 3T3-L1 cells showed statistically(P<0.05) higher GPDH activity in the single-culture than the co-culture, whereas CK results of L6 cells were vice versa(P<0.05). Overall, present results may indicate that co-culture system is more reliable and precise technique compared to single-culture. Further studies on several co-culture trials including different media conditions, supplementation of differentiating substances, molecular biological analysis, etc. should be required to obtain practical and fundamental mass data.

Fabrication of Cell Spheroids for 3D Cell Culture and Biomedical Applications

박세연,홍혜진,이현종 한국바이오칩학회 2023 BioChip Journal Vol.17 No.1

The significance of 3D cell culture—i.e., the maintenance of cell characteristics by mimicking the natural environment—has been increasing, and various 3D cell culture methods have been proposed. 3D cell culture is a widely used cell spheroid culture method, which is a representative method of matrix-free 3D cell culture. In contrast with that in conventional 2D cell culture, cells aggregate in a spherical shape with abundant cell–cell and cell–matrix interactions that more closely resemble the in vivo environment in a 3D culture. In a cell spheroid culture, the viability and functionality of spheroids change based on the cell type and morphology, and their controllable characteristics differ depending on the fabrication method used. Moreover, the mass production and size controllability of spheroids are dependent on the fabrication method used;therefore, the spheroid utilization method is also different. In this review, representative cell spheroid fabrication methods are examined, and the possibility of standardization is discussed so that these methods can be widely used. In addition, the biomedical applications of spheroids and their potential for future development are explored.

Evaluation of the osteogenic differentiation of gingiva-derived stem cells grown on culture plates or in stem cell spheroids: Comparison of two- and three-dimensional cultures

Lee, Sung-Il,Ko, Youngkyung,Park, Jun-Beom D.A. Spandidos 2017 Experimental and therapeutic medicine Vol.14 No.3

<P>Three-dimensional cell culture systems provide a convenient <I>in vitro</I> model for the study of complex cell-cell and cell-matrix interactions in the absence of exogenous substrates. The current study aimed to evaluate the osteogenic differentiation potential of gingiva-derived stem cells cultured in two-dimensional or three-dimensional systems. To the best of our knowledge, the present study is the first to compare the growth of gingiva-derived stem cells in monolayer culture to a three-dimensional culture system with microwells. For three-dimensional culture, gingiva-derived stem cells were isolated and seeded into polydimethylsiloxane-based concave micromolds. Alkaline phosphatase activity and alizarin red S staining assays were then performed to evaluate osteogenesis and the degree of mineralization, respectively. Stem cell spheroids had a significantly increased level of alkaline phosphatase activity and mineralization compared with cells from the two-dimensional culture. In addition, an increase in mineralized deposits was observed with an increase in the loading cell number. The results of present study indicate that gingiva-derived stem cell spheroids exhibit an increased osteogenic potential compared with stem cells from two-dimensional culture. This highlights the potential of three-dimensional culture systems using gingiva-derived stem cells for regenerative medicine applications requiring stem cells with osteogenic potential.</P>