ArticlePDF Available

Fetal bovine serum (FBS): Past – present – future

August 2017
ALTEX 35(1)

DOI:10.14573/altex.1705101

Authors:

J.B.F. Van der Valk

Utrecht University

Karen Bieback

Universität Heidelberg

Show all 17 authorsHide

The supplementation of culture medium with fetal bovine serum (FBS, also referred to as 'fetal calf serum') is still common practice in cell culture applications. Due to a number of disadvantages in terms of quality and reproducibility of in vitro data, animal welfare concerns, and in light of recent cases of fraudulent marketing, the search for alternatives and the development of serum-free medium formulations gained global attention. Here, we report on the 3rd Workshop on FBS, Serum Alternatives and Serum-free Media, where (a) regulatory aspects, (b) the serum dilemma, (c) alternatives to FBS, (d) case-studies of serum-free in vitro applications, and (e) the establishment of serum-free databases, were discussed. The whole process of obtaining blood from a living calf fetus to using the FBS produced from it for scientific purposes is de facto not yet legally regulated, despite the existing EU-Directive 2010/63/EU on the use of animals for scientific purposes. Together with above mentioned challenges, several strategies have been developed to reduce or replace FBS in cell culture media in terms of the 3Rs (Refinement, Reduction, Replacement). Most recently, releasates of activated human donor thrombocytes (human platelet lysates) have been shown to be one of the most promising serum alternatives when chemically defined media are not yet an option. Additionally, new developments in cell-based assay techniques, advanced organ-on-chip and microphysiological systems are covered in this report. Chemically-defined serum-free media are shown to be the ultimate goal for the majority of culture systems, and examples are discussed.

Scanning electron micrograph of resting (left) and activated (right) human thrombocytes, immobilized on sterile filters When platelets are activated, they undergo characteristic morphological changes and form long pseudopodia to adhere to the sites of vessel wall injury. In addition, thrombocytic granule factors are released by exocytosis: vasoactive peptides, factors for initiating and maintaining the clotting cascade, and growth factors for subsequent wound healing. Filters from Millipore Inc., pore size 0.22 µm. Micrograph taken by G. Gstraunthaler.

…

Test for the eye irritation potential of seven dilutions of sodium dodecyl sulphate toward L929 fibroblasts A: extracellular acidification rate (EAR), B: modulus of impedance (ІZІ)). The first five recordings of the basal rates are shown, followed by seven dilutions of sodium dodecyl sulphate.

…

Test for the eye irritation potential of seven dilutions of sodium dodecyl sulfate toward L929 fibroblasts Left: extracellular acidification rate (EAR), right: modulus of impedance (IZI). The first five recordings of the basal rates are shown, followed by seven dilutions of sodium dodecyl sulfate.

…

Schematic view of a 4-organ microfluidic platform (a) Schematic view of the microfluidic platform showing the different cell compartments of a 4-organ system. The system contained two holders for the separate culture devices. Total fluid volume was approximately 4 mL between the chambers and reservoirs. The sizes of the culture compartments were 35.8 x 18.4 x 0.3 mm for Chambers 1, 2, 3 and 29.8 x 15.4 x 0.7 mm for Chambers 4 and 5. The connecting channel dimensions were 5.7 x 1 x 0.3 mm. (b) Shear stress distribution in each compartment of the system. Reproduced with permission from (Oleaga et al., 2016).

…

Figures - uploaded by the authors

Content may be subject to copyright.

Content uploaded by Gerhard Gstraunthaler

Content may be subject to copyright.

Content uploaded by Gerhard Gstraunthaler

Content may be subject to copyright.

Content uploaded by J.B.F. Van der Valk

Content may be subject to copyright.

A preview of the PDF is not available

ALTEX 35 99 2018

Data

January 2018

J.B.F. Van der Valk · Karen Bieback · Christiane Buta · Brett Cochrane · Gerhard Gstraunthaler

Download

Fetal Bovine Serum (FBS): Past - Present - Future; ALTEX 35(1): 99-118 (2018)

Data

January 2018

J.B.F. Van der Valk · Karen Bieback · Christiane Buta · Brett Cochrane · Gerhard Gstraunthaler

Download

Modelling Meningioma Using Organoids: A Review of Methodologies and Applications

Article

Full-text available

Dec 2023

Meningiomas are the most common tumours of the central nervous system. According to the World Health Organization (WHO), this disease is classified into three different grades: 80% of meningioma patients present with benign grade I tumours, while less than 2% present with malignant grade III meningiomas. Despite affecting thousands of people worldwide, much remains unknown about this disease, and the development of systemic treatments is still far behind in comparison to other types of tumours. Therefore, forming 3D structures (spheroids and organoids) could facilitate research on the mechanisms of formation, proliferation, migration, and invasion of these, for the most part, benign tumours, while also helping in the process of drug development. To date, there are three published methods for the formation of meningioma organoids primarily derived from patient tissue samples. Organoids offer many advantages in the development of treatments because they recapitulate the cellular complexity within tumours. These new methodological advances could open a substantial number of possibilities for the further characterisation and treatment of meningiomas. This review includes an overview of the disease and a description and comparison of established protocols for meningioma organoid formation.

Induction of mesenchymal stem cell (MSC) differentiation from iPS cells using MSC medium

Article

Jan 2024

Mesenchymal stem cells (MSCs) and induced pluripotent stem (iPS) cells have great potential as cell sources for tissue engineering and regenerative medicine. This study aimed to investigate whether iPS cells can be differentiated into MSCs using MSCGM, a commercially available MSC culture system. The cells were characterized by flow cytometry, immunostaining, and gene expression analyses. We also examined their potential to differentiate into osteoblasts and chondrocytes. Our results showed that iPS cells cultured in MSCGM (iPS-MSCGM) exhibited a fibroblast-like morphology and expressed CD73 and CD90 genes, as well as positive markers for CD73, CD90, and CD105. Moreover, iPS-MSCGM cells demonstrated the ability to differentiate into osteoblasts and chondrocytes in vitro. This study demonstrates a new and simple method for inducing the differentiation of iPS cells to MSCs using MSCGM.

Alternative protein innovations and challenges for industry and consumer: an initial overview

Article

Full-text available

Nov 2023

Over one fourth of today's greenhouse gas emissions are the result of agriculture, with the production of meat representing a large portion of this carbon footprint. As the wealth of low- and middle-income countries continues to increase, the demand for animal-sourced protein, such as dairy and meat products, will escalate. At this point in time, livestock feed alone utilizes almost 40% of the world's cropland. The rapidly increasing world population, coupled with a need for environmental sustainability, has renewed our attention on animal-protein substitutes. Apprehensions over climate change have aided an acceleration in the research and development of alternative proteins, which may replace some animal-sourced protein over time. The alternative dairy and meat industry is developing at a yearly rate of 15.8% and is predicted to reach 1.2 trillion $USD by 2030. This emerging market incorporates new technologies in plant-made protein production, manufacturing of animal proteins by fermentation using microbial bioreactors, and accelerated production of cultivated (also known as cell-based) meat. These new technologies should change the global market drammatically. This article describes the history of the alternative protein industry and its' current status, then offers predictions of future pathways for this rapidly accelerating market. More speculatively, it discusses factors that lead to shifts in consumer behavior that trend toward the adoptation of new technologies.

Performance Evaluation of SARS-CoV-2 Viral Transport Medium Produced by Bangladesh Reference Institute for Chemical Measurements

Article

Full-text available

May 2023

Citation: Razu, M.H.; Monir, B.B.; Moniruzzaman, M.; Sarkar, S.; Akhter, S.; Kamal, S.; Hasan, M.A.; Afroze, M.; Imam, K.M.S.U.; Khan, M. Abstract: A viral transport medium (VTM) was developed following the Centers for Disease Control and Prevention, USA (US-CDC) standard operating procedure (SOP) DSR-052-05 with necessary improvisation and was used for storing coronavirus disease 2019 (COVID-19) swab specimens. Considering Bangladesh's supply chain and storage conditions, improvisation was essential for extending sample storage time while retaining efficiency. In-house VTM was produced using Hank's balanced salt solution (HBSS) supplemented with 1% bovine serum albumin V (BSA), 0.5 µg /mL of gentamicin sulfate, and 100 µg/mL of fluconazole. The produced VTM composition, quality, sterility, specificity, and efficiency were verified in-house and through an independent contract research organization (CRO). An accelerated stability study projected that under the recommended temperature (4 • C), it would remain stable for four months and preserve samples for over a month. The real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) test detected the targeted N gene and ORF1ab gene from the VTM stored samples. Our VTM is equally as effective as the Sansure Biotech VTM in keeping SARS-CoV-2 RNA specimens detectable in rRT-PCR (100% sensitivity and specificity in random and blinded samples). In conclusion, the BRiCM VTM will make the battle against pandemics easier by effectively collecting and storing nasopharyngeal and oropharyngeal swabs for COVID-19 detection.

Developmental, cytogenetic and epigenetic consequences of removing complex proteins and adding melatonin during in vitro maturation of bovine oocytes

Article

Full-text available

Oct 2023

Background In vitro maturation (IVM) of germinal vesicle intact oocytes prior to in vitro fertilization (IVF) is practiced widely in animals. In human assisted reproduction it is generally reserved for fertility preservation or where ovarian stimulation is contraindicated. Standard practice incorporates complex proteins (CP), in the form of serum and/or albumin, into IVM media to mimic the ovarian follicle environment. However, the undefined nature of CP, together with batch variation and ethical concerns regarding their origin, necessitate the development of more defined formulations. A known component of follicular fluid, melatonin, has multifaceted roles including that of a metabolic regulator and antioxidant. In certain circumstances it can enhance oocyte maturation. At this stage in development, the germinal-vesicle intact oocyte is prone to aneuploidy and epigenetic dysregulation. Objectives To determine the developmental, cytogenetic and epigenetic consequences of removing CP and including melatonin during bovine IVM. Materials and methods The study comprised a 2 x 2 factorial arrangement comparing (i) the inclusion or exclusion of CP, and (ii) the addition (100 nM) or omission of melatonin, during IVM. Cumulus-oocyte complexes (COCs) were retrieved from stimulated cycles. Following IVM and IVF, putative zygotes were cultured to Day 8 in standard media. RNAseq was performed on isolated cumulus cells, cytogenetic analyses (SNP-based algorithms) on isolated trophectoderm cells, and DNA methylation analysis (reduced representation bisulfite sequencing) on isolated cells of the inner-cell mass. Results Removal of CP during IVM led to modest reductions in blastocyst development, whilst added melatonin was beneficial in the presence but detrimental in the absence of CP. The composition of IVM media did not affect the nature or incidence of chromosomal abnormalities but cumulus-cell transcript expression indicated altered metabolism (primarily lipid) in COCs. These effects preceded the establishment of distinct metabolic and epigenetic signatures several days later in expanded and hatching blastocysts. Conclusions These findings highlight the importance of lipid, particularly sterol, metabolism by the COC during IVM. They lay the foundation for future studies that seek to develop chemically defined systems of IVM for the generation of transferrable embryos that are both cytogenetically and epigenetically normal.

Simultaneous detection of CA-125 and mesothelin by gold nanoparticles in surface plasmon resonance

Article

Nov 2023

Proliferation of mammalian cells with Chlorococcum littorale algal compounds without serum support

Article

Oct 2023
BIOTECHNOL PROGR

In recent years, serum-free medium for mammalian cell cultivation has attracted a lot of attention, considering the high cost of production and environmental load involved in developing the conventional animal sera. The use of alternative growth-promoting products in mammalian cell cultivation such as extracts from microalgae has proven to be quite beneficial and environmental-friendly. This research aims to cultivate mammalian cells with growth-promoting factors derived from Chlorococcum littorale. We have established a simple extraction using the ultrasonication method and applied the extract in place of serum on mammalian C2C12 cell lines, 3T3 cell lines, and CHO cell lines to compare and analyze the effectiveness of the extract. Cell passage was conducted in a suspended culture condition with the addition of the extract. The results indicate that the extract from microalgae shows a high proliferation rate in all cell lines without fetal bovine serum. Moreover, it is eco-friendly and has huge potential to replace the traditional cell culture system. It could be applied in the fields of regenerative medicine, gene/cell therapies, as well as cultured meat production.

Addressing Key Questions in Organoid Models: Who, Where, How, and Why?

Article

Full-text available

Nov 2023
INT J MOL SCI

Organoids are three-dimensional cellular structures designed to recreate the biological characteristics of the body’s native tissues and organs in vitro. There has been a recent surge in studies utilizing organoids due to their distinct advantages over traditional two-dimensional in vitro approaches. However, there is no consensus on how to define organoids. This literature review aims to clarify the concept of organoids and address the four fundamental questions pertaining to organoid models: (i) What constitutes organoids?—The cellular material. (ii) Where do organoids grow?—The extracellular scaffold. (iii) How are organoids maintained in vitro?—Via the culture media. (iv) Why are organoids suitable in vitro models?—They represent reproducible, stable, and scalable models for biological applications. Finally, this review provides an update on the organoid models employed within the female reproductive tract, underscoring their relevance in both basic biology and clinical applications.

Optimizing SH-SY5Y Cell Culture: Exploring the Beneficial Effects of an Alternative Media Supplement on Cell Proliferation and Viability

Preprint

Full-text available

Oct 2023

In the quest to unravel the mysteries of neurological diseases, comprehending the underlying mechanisms is supreme. The SH-SY5Y human neuroblastoma cell line serves as a crucial tool in this endeavor; however, the cells are known for its sensitivity and slow proliferation rates. Typically, this cell line is cultured with 10% Fetal Bovine Serum (FBS) supplement. Nu-Serum (NuS), a low-protein alternative to FBS, is promising to advance cell culture practices. Herein, we evaluated the substitution of NuS for FBS to test the hypothesis that an alternative serum supplement can aid and promote SH-SY5Y cell proliferation and differentiation. Our findings revealed that the NuS-supplemented group exhibited a notable increase in adhered cells compared to both the FBS and serum-free (SF) groups. Importantly, cell viability remained high in both sera treated groups, with the NuS-supplemented cells displaying significantly larger cell sizes compared to the SF-treated group. Furthermore, cell proliferation rates were higher in the NuS-treated group, and neuroblast-like morphology was observed earlier than FBS group. Notably, both FBS and NuS supported the differentiation of these cells into mature neurons. Our data supports NuS as an alternative for SH-SY5Y cell culture, with the potential to elevate the quality of research in the neuroscience field.

Serum albumin maintains Wnt water-solubility and activity

Preprint

Full-text available

Sep 2023

Wnt proteins regulate adult tissue homeostasis and repair by driving stem cell self-renewal and differentiation. High-performance Wnt preparations have enormous therapeutic potential, especially alongside various stem cell technologies. Currently, most of these Wnt preparations contain Fetal Bovine Serum or the detergent CHAPS to maintain Wnt solubility and activity. Recently, afamin was identified as a serum factor that solubilizes Wnt3a in conditioned media, obviating the requirement for animal sera. Here, we report serum albumin is required for afamin-mediated solubilization of Wnt3a in conditioned media. Moreover, Serum Albumin-mediated solubilization of purified Wnt3a in tubes does not require afamin. This means conventional CHAPS-Wnt3a preparations can be modified into Serum Albumin-purified Wnt3a preparations by exchanging CHAPS for Serum Albumin through dialysis. Serum Albumin-purified Wnt3a preparations effectively promote the growth of human stem cell organoids. These data suggest Serum Albumin as a physiological factor for maintaining Wnt3a activity in therapeutic applications.

Chemisch definiert – ein zellbasierter Zytotoxizitätsassay ohne fötales Kälberserum

Article

Full-text available

Feb 2017

Joachim Wiest

Tests for cytotoxicity–respectively biocompatibility–are performed e. g. for biological evaluation of medical devices or during the development of microphysiometric systems. Here, often cell culture methods which involve the use of fetal bovine serum (FBS) are employed. However, using FBS raises scientific and ethical concerns. In the presented work a protocol for a chemically defined test for cytotoxicity is described.

Human Platelet Lysates as a Serum Substitute in Renal Epithelial Cell Culture

Article

Apr 2012

Cultured renal epithelia form monolayers of differentiated, polarized cells. On permeable supports, an apical and a basolateral compartment are separated by the cultured epithelium to study epithelial transport in vitro. However, culture conditions and culture media have a significant impact. In this respect, the quality of fetal bovine serum (FBS) seemed to be of major importance. The high lot‐to‐lot variablity of FBS was found to substantially influence the differentiation of epithelial cultures with regard to the generation of a transepithelial electrical resistance (TEER). Thus, batch‐testing of FBS was required. We recently reported on the use of human platelet lysates (PL) as a replacement for FBS. The growth promoting capacity of PL was tested on renal epithelial cell lines, for which differentiation end points are well established. PL support growth, proliferation and differentiation of LLC‐PK1, HK‐ 2 and MDCK cells. In addition, TEER was monitored in filter‐grown LLC‐PK1 and in MDCK II and MDCK I epithelia. Low‐resistance epithelia generated a TEER of 150–250 Ω•cm2 in PL‐media, as seen with FBS, and high‐resistance MDCK I retained their TEER of 8,000 Ω•cm2. PL are a valuable, animal‐derived component‐free substitute for FBS in renal epithelial cell culture with a proven high batch‐to batch uniformity.

Confluent Trends in Cell Culture Media

Article

Feb 2016

Angelo DePalma

Zell- und Gewebekultur

Book

Jan 2013

Eye Banking in the 21st Century: How Far Have We Come? Are We Prepared for What’s Ahead?

Article

Sep 2012

PURPOSE: This review of milestones in eye banking describes efforts by the eye banking community to ensure the quality and quantity of corneal tissue over the last century and anticipates key challenges going forward. METHODS: This account draws on a review of the scientific literature, public documents, and eye banking statistics and is augmented by the recollection of the author, discussions with eye bankers, and an analysis of pressing medical and nonmedical issues that will bear on the future success of eye banking in the United States and internationally. RESULTS: The author identifies five eras of eye banking, highlighting scientific breakthroughs in surgical techniques, storage, and instrumentation; the professionalization of eye banks through development of medical standards and accreditation programs; and the advent of laws and regulations leading to reimbursement changes and donor legislation. The author next identifies five strengths exhibited by the community to achieve those milestones and delineates crucial questions posed by an ever-expanding array of unprecedented demographic, socioeconomic, geopolitical, biological, regulatory, technological, cultural, and ethical challenges. CONCLUSIONS: Technological advances and collaborative efforts have brought the eye banking community to the enviable position it enjoys today. Only by building on its historical strengths can eye banking professionals worldwide successfully evolve their role in an increasingly complex future.

Towards chemically‐defined, serum‐free media for mammalian cell culture

Article

Jan 1986

HR. Maurer

A consensus introduction to serum replacements and serum-free media for cellular therapies

Article

Dec 2016
CYTOTHERAPY

The cell therapy industry is a fast-growing industry targeted toward a myriad of clinical indications. As the cell therapy industry matures and clinical trials hit their pivotal Phase 3 studies, there will be a significant need for scale-up, process validation, and critical raw material quality assurance. Part of the well discussed challenges of upscaling manufacturing processes there is a less discussed issue relating to the availability of raw materials in the needed quality and quantities. The FDA recently noted that over 80% of the 66 investigational new drug (IND) applications for mesenchymal stem cell (MSC) products analyzed described the use of FBS during manufacturing. Accumulated data from the past years show an acceleration in serum consumption by at least 10%-15% annually, which suggests that the global demand for serum may soon exceed the supply. Ongoing concerns of safety issues due to risks of various pathogen contaminations, as well as issues related to the aforementioned serum variability that can affect final product reproducibility, are strong motivators to search for serum substitutes or serum-free media. it is important to note that there are no accepted definitions for most of these terms which leads to misleading's and misunderstandings, where the same term might be defined differently by different vendors, manufacturer, and users. It is the drug developer's responsibility to clarify what the supplied labels mean and to identify the correct questions and audits to ensure quality. The paper reviews the available serum replacements, main components, basic strategies for replacement of serum and suggests definitions.

Influence of Donor Age and Stimulation Intensity on Osteogenic Differentiation of Rat Mesenchymal Stromal Cells in Response to Focused Low-Intensity Pulsed Ultrasound

Article

Sep 2016

A focused low-intensity pulsed ultrasound (FLIPUS) was used to investigate the effects of stimulation period, acoustic intensity and donor age on the osteogenic differentiation potential of rat mesenchymal stromal cells (rMSCs). rMSCs from 3- and 12-mo-old female Sprague Drawly rats were isolated from bone marrow and stimulated 20 min/d with either 11.7 or 44.5 mW/cm(2) (spatial average temporal average intensity) for 7 or 14 d. Osteogenic differentiation markers, i.e., Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and degree of matrix calcification were analyzed. On day 7 of stimulation, OCN gene expression was enhanced 1.9-fold in cells from young rats when stimulated with low intensity. The low intensity also led to a 40% decrease in RUNX2 expression on day 7 in aged cells, whereas high intensity enhanced expression of RUNX2 on day 14. FLIPUS treatment with low intensity resulted in a 15% increase in extracellular matrix mineralization in young but not old rMSCs. These differences suggest the necessity of a donor-age related optimization of stimulation parameters.

Regulation (EC) No 1907/2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Article

Jan 2006

EC (European Commission

Reproducibility: Respect your cells!

Article

Sep 2016

Monya Baker

p>Numerous variables can torpedo attempts to replicate cell experiments, from the batch of serum to the shape of growth plates. But there are ways to ensure reliability.</p