Research on Fetal Bovine Serum substitutes obtained from porcine slaughter by-product for cell culture

다국어 초록 (Multilingual Abstract)

Research on Fetal Bovine Serum substitutes obtained from porcine slaughter by-product for cell culture Juhyun Lee Major in Animal Resource Biotechnology Department of Animal Science & Biotechnology The Graduate School Chung-Ang University Fetal Bovine Serum(FBS) contains various nutrients and is essential for cell culture. However, due to the high price and ethical issues associated with obtaining it, research is continuously being conducted to develop materials that can replace FBS. Domestic slaughter by-products are high in nutrients and production is increasing every year, but most of them are discarded because they cannot be fully used. Therefore, the purpose of this study is to develop an FBS substitute for cell culture derived from slaughter by-products, which are mostly discarded after slaughter. Pig intestine by-products were extracted with enzymes and used in experiments, and blood from cows, pigs, and chickens was centrifuged to produce enzyme digests and serum, respectively. A combination of serum and enzymatic digests has been used to replace FBS for the propagation of cell lines and microsatellite cells. Additionally, experiments were conducted to determine whether muscle cells affected the differentiation of myotubes. FBS substitution experiments on various cells showed that FBS was successfully replaced in terms of cell morphology, proliferation, and differentiation. In this study, we discovered the potential and effectiveness of FBS substitutes in slaughter by-products in cell culture. The method used in this study may contribute to the development of additional FBS substitutes by exploiting the potential of various by-products.

국문 초록 (Abstract)

소 태아 혈청(Fetal Bovine Serum)는 다양한 영양소를 함유하고 있어 세포 배양에 필수적으로 사용된다. 그러나 높은 가격과 이를 획득하는데 따른 윤리적 문제로 인해 FBS 를 대체할 수 있는 물질 개발이 지속적으로 연구가 진행되고 있다. 국내의 도축 부산물은 높은 영양소를 가지고 있으며 매년 생산량이 늘어나고 있으나 전부 이용되지 못해 대부분 폐기되고 있다. 따라서 본 연구의 목적은 도축 후 대부분 폐기되는 도축 부산물로부터 유래된 세포 배양용 FBS 대체물질을 개발하는 것이다. 돼지 내장 부산물을 효소 추출하여 실험에 사용하였고, 소, 돼지, 닭의 혈액을 원심분리하여 각각 효소 분해물과 혈청을 생성하였다. 혈청과 효소 분해물의 조합은 세포주 및 소 위성 세포의 증식을 위해 FBS 를 대체하는 데 사용되었습니다. 추가적으로 근육세포에서는 근관의 분화에 영향을 주는지 확인하는 실험을 진행하였다. 다양한 세포주에 대한 FBS 대체실험은 세포의 형태 및 증식과 분화에서 성공적으로 FBS 를 대체하는 결과가 나타났습니다. 이 연구에서 우리는 세포 배양의 도축 부산물에서 FBS 대체물의 잠재력과 효과를 발견했습니다. 본 연구에서 사용된 방법은 다양한 부산물의 잠재력을 활용하여 추가적인 FBS 대체품 개발에 기여할 수 있습니다.

목차 (Table of Contents)

• Literature review 1
• I. Porcine by-product production and consumption 1
• II. Definition of slaughter by-products 4
• II.1. Visceral by-products 10
• II.2. Slaughter blood 14
• III. Research of cell culture 16
• III.1. General information about Fetal Bovine Serum(FBS) 21
• III.2. Experiment about FBS replacement 25
• 제1장. Introduction 31
• 제2장. Materials and Methods 33
• 2.1. Analysis of porcine by-product composition 33
• 2.1.1. Method of obtain FBS substitute from slaughter by-product 33
• 2.1.2. Protein composition analysis using SDS-PAGE 37
• 2.1.3. Analysis of amino acid 38
• 2.2. Cell culture 39
• 2.3. Cell experiment with FBS substitute 39
• 2.3.1. Methdod of cell line culture 42
• 2.3.2. Methdod of animal cell culture 42
• 2.4. Analysis of cell viability using MTT assay 43
• 2.5. Comparison of cell differentiation rates using fluorescent staining 43
• 2.5.1. Methdod of immunocytochemistry 44
• 2.6. Statistical analysis 45
• 제3장. Results and discussion 46
• 3.1. Analysis of enzymatic hydrolysis of porcine by-product composition 46
• 3.1.1. Protein quantitation of enzymatic hydrolysate by-product 46
• 3.1.2. Analysis of amino acid 51
• 3.2. Cell experiment with addition of FBS substitute 55
• 3.3. Cell culture experiments using FBS substitute 57
• 3.3.1. Bovine satellite cell 57
• 3.3.2. C2C12 cell 60
• 3.3.3. HepG2 cell 62
• 3.3.4. HF cell 64
• 3.3.5. RAW 264.7 cell 66
• 3.3.6. STO cell 68
• 3.4. Cell differentiation with FBS substitute 70
• 제4장. Conclusion 74
• Reference 78
• 국문초록 92