Identification and Quantification of Proteins from Preparative Partition Chromatography and Peptides from Organic Extraction of Fetal Versus Adult Bovine Serum using Nano-Spray-LC-ESI-MS/MS

10.32920/ryerson.14645256 ◽

2021 ◽

Author(s):

Zhuo Zhen Chen

Keyword(s):

Growth Factors ◽

Bovine Serum ◽

Fetal Bovine Serum ◽

Intact Proteins ◽

Endogenous Peptides ◽

Fetal Serum ◽

Adult Bovine Serum ◽

Alpha Feto Protein ◽

Fetal Bovine ◽

Insulin Like Growth Factors

Blood proteins communicate with many different cells, tissue and organs; perform key functions in the immune system and may be of particular biological complexity. One of the most widely used blood products in the laboratory is fetal bovine serum for cell culture. There are ethical and practical concerns regarding the use of fetal serum from animals and alternative serum-free replacements have been attempted using platelet lysates. Previous biochemical experiments have shown that FBS apparently contained factors such as alpha-feto protein (AFP) and insulin-like growth factors that may support the indefinite cell growth and division of certain cell lines. It is presumed that a set of as yet undefined growth factors transform cells growth resulting in rapid proliferation. Cultured Raw cells 264.7 in adult bovine serum multiplied slowly and differentiated into elongated cells with a dendritic shape, which died after the first few generations. On the contrary, in fetal bovine serum, cultured cells multiplied rapidly and formed many smaller cells with a rounded shape through many cell passages. Three independent batches of fetal bovine serum were tested on Raw cells 264.7 macrophages to confirm that they supported cell growth in culture compared to three independent batches of adult bovine serum. The intact proteins of each serum sample were separated by partition chromatography into 16 fractions with an increasing step gradient of salts over quaternary amine resin (proteomics). The endogenous peptides were precipitated with 90% of acetonitrile and extracted into 10 fractions with a decreasing step gradient of acetonitrile in water (peptidomics). Trypsin digested intact proteins and endogenous peptides were then analyzed on a fresh C18 nano-HPLC column with random and independent sampling by LC-ESI-MS/MS. The fractionated mass spectra were identified with SEQUEST and X!TANDEM algorithms. Redundant use of MS/MS spectra were flirted out with the SQL Server system and the R statistical analysis system was used to perform Chi Square (X2) analysis of frequency counts and ANOVA of the log10 precursor intensity results. Alpha-feto protein, fetal albumin, insulin, insulin like growth factors, platelet derived growth factors and proteins associated with HRAS/AKT growth pathway at the level of ligand, receptors, receptor associated enzyme and nucleic acid binding proteins including transcription factors were observed to be specifically enriched in fetal serum.

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Biopotency of fetal bovine serum, and insulin and insulin-like growth factors I and II in enhancing whole-body protein synthesis of chicken embryos cultured in vitro

Comparative Biochemistry and Physiology Part C Pharmacology Toxicology and Endocrinology ◽

10.1016/0742-8413(95)00049-t ◽

1995 ◽

Vol 111 (2) ◽

pp. 281-286 ◽

Cited By ~ 3

Author(s):

Tatsuo Muramatsu ◽

Reinhard Pinontoan ◽

Jun-ichi Okumura

Keyword(s):

Protein Synthesis ◽

Growth Factors ◽

Bovine Serum ◽

Fetal Bovine Serum ◽

Whole Body ◽

Body Protein ◽

Chicken Embryos ◽

Fetal Bovine ◽

Insulin Like Growth Factors

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Insulin-like growth factors I and II in fetal and adult bovine serum. Purification, primary structures, and immunological cross-reactivities.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)36130-6 ◽

1986 ◽

Vol 261 (2) ◽

pp. 569-575

Author(s):

A Honegger ◽

R E Humbel

Keyword(s):

Growth Factors ◽

Bovine Serum ◽

Adult Bovine Serum ◽

Primary Structures ◽

Insulin Like Growth Factors

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Clinical grade cultivation of human Schwann cell, by the using of human autologous serum instead of fetal bovine serum and without growth factors

Cell and Tissue Banking ◽

10.1007/s10561-011-9250-8 ◽

2011 ◽

Vol 13 (2) ◽

pp. 281-285 ◽

Cited By ~ 11

Author(s):

Hamid-Reza Aghayan ◽

Babak Arjmand ◽

Abbas Norouzi-Javidan ◽

Hooshang Saberi ◽

Masoud Soleimani ◽

...

Keyword(s):

Growth Factors ◽

Schwann Cell ◽

Bovine Serum ◽

Fetal Bovine Serum ◽

Autologous Serum ◽

Clinical Grade ◽

Fetal Bovine

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Putrescine-oxidase activity in adult bovine serum and fetal bovine serum

In Vitro ◽

10.1007/bf02618948 ◽

1979 ◽

Vol 15 (4) ◽

pp. 252-257 ◽

Cited By ~ 8

Author(s):

William A. Gahl ◽

Henry C. Pitot

Keyword(s):

Bovine Serum ◽

Oxidase Activity ◽

Fetal Bovine Serum ◽

Adult Bovine Serum ◽

Fetal Bovine ◽

Putrescine Oxidase

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Comparison of Human Platelet Lysate versus Fetal Bovine Serum for Expansion of Human Articular Cartilage–Derived Chondroprogenitors

Cartilage ◽

10.1177/1947603520918635 ◽

2020 ◽

pp. 194760352091863 ◽

Cited By ~ 1

Author(s):

Upasana Kachroo ◽

Shikha Mary Zachariah ◽

Augustine Thambaiah ◽

Aleya Tabasum ◽

Abel Livingston ◽

...

Keyword(s):

Growth Factors ◽

Bovine Serum ◽

Human Platelet ◽

Fetal Bovine Serum ◽

Surface Marker ◽

Platelet Lysate ◽

Surface Marker Expression ◽

Trilineage Differentiation ◽

Human Platelet Lysate ◽

Fetal Bovine

Purpose Articular chondroprogenitors, a suitable contender for cell-based therapy in cartilage repair, routinely employ fetal bovine serum (FBS) for expansion and differentiation. The possibility of transplant rejections or zoonoses transmissions raise a need for xeno-free alternatives. Use of human platelet lysate (hPL), a nutrient supplement abundant in growth factors, has not been reported for human chondroprogenitor expansion thus far. Our aim was to compare the biological profile of chondroprogenitors grown in hPL versus FBS. Methods Chondroprogenitors were isolated from 3 osteoarthritic knee joints. Following differential fibronectin adhesion assay, passage 0 cells grown in (a) 10% FBS and (b) 10% hPL were considered for assessment of growth kinetics, surface marker expression, gene expression, and trilineage differentiation. Latent transforming growth factor–β1 (TGFβ1) levels were also measured for each culture medium used. Results Cellular proliferation was significantly higher in cells grown with hPL ( P < 0.01). Surface marker expression was comparable except in CD-146 where hPL group had significantly higher values ( P = 0.03). Comparison of mRNA expression revealed notably low values of collagen I, collagen X, aggrecan, and collagen II ( P < 0.05). Trilineage differentiation was seen in both groups with higher alizarin red uptake noted in hPL. There were also significantly higher levels of latent TGFβ1 in the medium containing hPL as compared to FBS. Conclusions This is the first in vitro xeno-free study to affirm that hPL can serve as an optimal growth supplement for expansion of articular chondroprogenitors, although an in-depth assessment of resident growth factors and evaluation of different dilutions of hPL is required to assess suitability for use in translational research.

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PENGARUH LISAT PLATELET FASE SIKLUS MENSTRUASI TERHADAP AKTIVITAS FIBROBLAS KULIT MANUSIA

Media Dermato Venereologica Indonesiana ◽

10.33820/mdvi.v46i1.48 ◽

2019 ◽

Vol 46 (1) ◽

Author(s):

Thianti Sylviningrum ◽

Flandiana Yogianti ◽

Sunardi Radiono ◽

Yohanes Widodo Wirohadidjojo

Keyword(s):

Growth Factors ◽

Bovine Serum ◽

Fetal Bovine Serum ◽

Platelet Rich Fibrin ◽

Fetal Bovine

Lisat platelet dari platelet rich fibrin (PRF) adalah material berisi growth factors, estradiol (E2), dan leptin. Pada fase ovulasi (FO) dijumpai kadar E2, jumlah platelet, dan leukosit tertinggi, dimana interaksinya dapat menghasilkan growth factors yang meningkatkan aktivitas fibroblas dermis. Penelitian ini bertujuan untuk mengetahui efek lisat platelet dari PRF fase bleeding (FB) dan FO pada aktivitas fibroblas dermis.Biakan fibroblas dermis dibedakan menjadi 3 kelompok, biakan dalam media kultur dan fetal bovine serum (FBS) 1% sebagai kontrol negatif, biakan dalam media kultur dan FBS 10% sebagai kontrol positif. Kelompok perlakuan adalah biakan fibroblas yang diberi lisat platelet dari PRF FB dan FO 10%, 20%, dan 40% selama 72 jam.Perbedaan bermakna (p<0,05) dijumpai pada rerata indeks proliferasi antara kelompok kontrol positif dengan fibroblas terpajan lisat platelet dari PRF FB dan FO 20% dibandingkan dengan kontrol negatif, serta rerata densitas optik (DO) timbunan kolagen dari fibroblas yang diberi lisat platelet dari PRF FB dan FO 20% dibandingkan dengan semua kelompok kontrol. Tidak ada perbedaan bermakna (p>0,05) pada rerata indeks proliferasi dan DO timbunan kolagen antara kelompok fibroblas yang terpajan semua konsentrasi lisat platelet dari PRF FB dan FO. Peningkatan aktivitas fibroblas dermis oleh lisat platelet dari PRF FO tidak berbeda dibandingkan dengan lisat platelet dari PRF FB.Kata kunci : lisat platelet, platelet rich fibrin, fase bleeding, fase ovulasi, fibroblas, kolagen

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Human Cells Grown With or Without Substitutes for Fetal Bovine Serum

Cell Medicine ◽

10.1177/2155179018755140 ◽

2018 ◽

Vol 10 ◽

pp. 215517901875514 ◽

Cited By ~ 5

Author(s):

John E. Piletz ◽

Jennifer Drivon ◽

John Eisenga ◽

Will Buck ◽

Sabrina Yen ◽

...

Keyword(s):

Endothelial Cell ◽

Cell Line ◽

Bovine Serum ◽

Fetal Bovine Serum ◽

Neuronal Cell ◽

Pharmaceutical Products ◽

Endothelial Cell Line ◽

Adult Bovine Serum ◽

Human Platelet Lysate ◽

Fetal Bovine

Safety concerns over cell-derived pharmaceutical products being manufactured in supplements of fetal bovine serum (FBS) have ignited pleas to replace FBS. Herein, four newly marketed alternatives to FBS were compared: a xeno-free product called Cell-Ess®, a human platelet lysate marketed as GroPro®, and two mixtures of adult bovine serum varying in their proportions of neonatal growth factors, called Liporo® and FetalGro®. An endothelial cell line (C2BBe1) and a neuronal cell line (SHSY5Y) near confluency in media with 10% FBS were selectively scraped and taken through a 25-day step-wise algorithm to replace FBS, and another human endothelial cell line (HRA-19) was studied to replicate C2BBe1. Cells were stained, counted, and compared for viability, migration, and spheroids. The C2BBe1 and HRA-19 cell lines failed to proliferate in 10% Cell-Ess® but grew in 10% GroPro® or 10% FetalGro® reasonably well compared to reference 10% FBS. With SH-SY5Y, only FetalGro® approached FBS's efficacy. These were all inferior to 11 different branded lots of FBS (positive controls), but five days into switching just amongst the FBS brands, 4 of 11 supported less proliferation than reference FBS in endothelial HRA-19 ( p < 0.004). Moreover, neurospheres were enriched in two branded lots of FBS and FetalGro® (each p < 0.004), neurospheres being an unwanted phenotype for any neuronal cell application. Because platelet-derived GroPro® stood out amongst the non-FBS growth supplements to allow proliferation without inducing spheroids, it seems the best (mindful that the cells still grew slower in it compared to FBS). While no perfect replacement was found amongst the alternatives to FBS, the algorithm for switching should be useful in future testing of new alternatives to FBS as the need arises to switch from FBS and expand pharmaceutical products with safety for human use.

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