ReviewMethods for growth of cultured cells in serum-free medium
References (65)
- R.G. Ham et al.
- G. Sato
- J.P. Mather et al.
Exp. Cell Res
(1979) - J. Bottenstein et al.
- A. Vogt et al.
Exp. Cell Res
(1969) - M. Hook et al.
Biochem. Biophys. Res. Commun
(1977) - J. Orly et al.
Cell
(1979) - S. Strickland et al.
Cell
(1978) - A. Rizzino et al.
Exp. Cell Res
(1979) - K. Higuichi
Advan. Appl. Microbiol
(1973)
In Vitro
In Vitro
Nature New Biol
Science
Nature (London)
J. Exp. Med
J. Cell Biol
Nature (London)
In Vitro
Nature (London)
Nature (London)
In Vitro
Cancer Res
Cited by (827)
Trace metals in cellular metabolism and their impact on recombinant protein production
2021, Process BiochemistryReplacement of serum and increasing use of chemically defined media demands optimisation of trace metal components for biomanufacturing applications. Trace metal availability can impact culture performance, productivity and product quality. Several trace metals are cofactors of metabolic and other enzymes, and thus their availability regulates cellular metabolism. Additionally, they can also affect the availability of other trace metals and stability of some medium components. Such factors also need to be considered while formulating trace metal concentrations in the culture medium. Due to their very low concentrations, these components are susceptible to substantial variability arising from contaminants from other raw material and leaching from process equipment and can contribute to process variability. Understanding the role and impact of trace metals will help develop strategies to achieve targeted process parameters and increase process robustness vis-à-vis any lot-to-lot variability in trace metal concentration in culture medium. This review describes the role of trace metals, particularly manganese, copper and zinc, in central carbon metabolism to aid in understanding the basis of metal-mediated effects on culture performance and provides a comprehensive review of the reported impact of trace metals on CHO cell culture performance and recombinant protein quality.
Effect of serum replacement on murine spermatogonial stem cell cryopreservation
2021, TheriogenologyCryopreservation of spermatogonial stem cells (SSCs) is a necessity to preserve the genetic information of valuable livestock herds and to produce transgenic animals. However, serum, a key component that allows efficient cryopreservation, has many limitations attributed to its undefined composition, inter-batch variations, and contamination potential. Therefore, we aimed to establish a method for serum-free cryopreservation of SSCs. To evaluate the cryopreservation efficiency of serum replacements, we assessed the recovery rate, relative proliferation potential, proliferation capacity, and apoptosis capacity. SSCs were characterized, and their functional activity was determined through immunofluorescence, RT-qPCR, and spermatogonial transplantation. The efficiency of each serum replacement was compared to that of the negative control (10% DMSO in DPBS) and positive control (10% DMSO and 40% FBS in DPBS). Our results indicated that cryopreservation with 5% human serum albumin (rHSA) exhibited a higher relative proliferation potential (274.0 ± 13.4%) than with DMSO control (100 ± 8.6%), with no significant difference from the 40% FBS (190.0 ± 20.1%). Moreover, early apoptosis also significantly decreased to a greater extent with 5% rHSA (5.1 ± 0.7%) than with DMSO control (12.9 ± 0.8%) and was at a level comparable to the 40% FBS (4.9 ± 0.8%). In addition, the SSCs cryopreserved with 5% rHSA exhibited normal self-renewal and differentiation abilities. In conclusion, 5% rHSA is a potential serum replacement for SSC cryopreservation, with properties comparable to that of serum. These results would contribute to the application of SSCs in improving livestock and in future clinical trials for human infertility treatment.
A novel serum free primary astrocyte culture method that mimic quiescent astrocyte phenotype
2019, Journal of Neuroscience MethodsPrimary astrocyte cultures have been used for decades to study astrocyte functions in health and disease. The current primary astrocyte cultures are mostly maintained in serum-containing medium which produces astrocytes with a reactive phenotype as compared to in vivo quiescent astrocytes. The aim of this study was to establish a serum-free astrocyte culture medium that maintains primary astrocytes in a quiescent state.
Serum free astrocyte base medium (ABM) supplemented with basic fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) (ABM-FGF2-EGF) or serum supplemented DMEM (MD-10%FBS) was used to culture primary astrocytes isolated from cerebral cortex of postnatal day 1 C57BL/6 mice.
Compared to astrocytes cultured in MD-10%FBS medium, astrocytes in ABM-FGF2-EGF had higher process bearing morphologies similar to in vivo astrocytes. Western blot, immunostaining, quantitative polymerase chain reaction and metabolic assays revealed that astrocytes maintained in ABM-FGF2-EGF had enhanced glycolytic metabolism, higher glycogen content, lower GFAP expression, increased glutamine synthase, and glutamate transporter-1 mRNA levels as compared to astrocytes cultured in MD-10% FBS medium.
These observations suggest that astrocytes cultured in ABM-FGF2-EGF media compared to the usual FBS media promote quiescent and biosynthetic phenotype similar to in vivo astrocytes.
This media provides a novel method for studying astrocytes functions in vitro under physiological and pathological conditions.
Pancreas and islet preservation
2019, Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas: Volume 1Pancreatic islets contain endocrine beta and alpha cells that produce insulin and glucagon, respectively, two hormones key to regulating blood sugar. Recent technological advances in human islet transplantation (ITx) have triggered renewed interest in whole organ and islet preservation methods aimed at protecting the structural and functional integrity of islets during the process of islet manufacturing, from the point of organ procurement to the point of islet infusion into the recipient. Islet isolation is a complex, multistep and laborious procedure influenced by numerous factors that require highly trained and skillful personnel. Due to a high islet attrition rate during isolation and limited islet viability after purification, most current ITx protocols require islets from more than one pancreas. The need for multiple donors represents a major economical and clinical barrier to expand the number of diabetic patients that could benefit from ITx. Ischemia and hypoxia have been identified as prominent factors contributing to islet losses during isolation and after ITx. Delivery of appropriate amounts of oxygen and other preservation measures must be implemented at the organ, tissue and cellular level to improve islet viability and preserve beta and alpha cell function. The current chapter reviews the mechanistic impact of ischemia and hypoxia on islet function and viability, as well as current methods of islet preservation prior, during and after islet isolation, purification, culture, and distribution.
Oxidative stress differentially impacts male and female bovine embryos depending on the culture medium and the stress condition
2018, TheriogenologyMale and female embryos are known to differ for their metabolism and response to environmental factors very early in development. The present study aimed to evaluate the response to oxidative stress of male and female bovine embryos at the morula-blastocyst stages in terms of developmental rates, total cell number and apoptotic rates in two culture conditions. Embryos where cultured in a medium supplemented with either 5% fetal calf serum (FCS) or 4 mg/mL bovine serum albumin and a mixture of insulin, transferrin and selenium (BSA-ITS). Oxidative stress was applied at Day-5 post insemination (pi) by adding either AAPH or menadione to the culture medium, and blastocysts were analyzed at Day-7pi. The impact on development and blastocyst quality was dependent on the culture medium and the stress inducer but differed between male and female embryos. Male embryos resisted better to oxidative stress in FCS supplemented medium, no matter the stress inducer. Accordingly, the impact on blastocyst cell number tended to be higher in female blastocysts after stress induction with AAPH in FCS supplemented medium. On the other hand, in BSA-ITS supplemented medium, female embryos were more resistant to AAPH induced stress, while menadione had no impact on sex ratio. The weaker resistance of males to AAPH in this medium is in accordance with their trend to show a higher increase in apoptotic rates than females in this condition. In conclusion, this study shows that oxidative stress has differential impact on male and female bovine blastocysts depending on the culture condition and on the way oxidative stress is induced.
Effect of cationic gemini surfactant and its monomeric counterpart on the conformational stability and esterase activity of human serum albumin
2018, Journal of Molecular LiquidsThe binding effect of gemini surfactant, hexanediyl-α,ω-bis-(N-(2-hydroxyethyl)–N-methylhexadecylammonium bromide) (16-6-16 MEA) and its monomeric counterpart N-(2-hyroxyethyl)-N,N-dimethylhexadecylammonium bromide (16-MEA) on the structure and esterase like activity of human serum albumin (HSA) was unravelled by using UV–visible, fluorescence, three dimensional fluorescence, time resolved fluorescence and circular dichroism techniques in combination with molecular docking and molecular dynamic simulation methods. Fluorescence quenching mechanism was employed to estimate the Stern–Volmer quenching constants, Ksv, and the corresponding thermodynamic parameters like ∆G, ∆S and ∆H for HSA-16 MEA/16-6-16 MEA systems. The results showed the static quenching mechanism and spontaneous binding of 16 MEA/16-6-16 MEA with HSA. In addition to other interactions, the hydrophobic interaction (which is more effective in case gemini surfactant) plays major role in the process of binding of both the surfactants with HSA. Synchronous fluorescence reveals that there is a slight effect of 16 MEA/16-6-16 MEA on the fluorescence intensity of tyrosine but a significant effect on tryptophan. UV–visible results obtained, suggest the complex formation between HSA and 16MEA/16-6-16 MEA through static quenching mechanism. Circular dichroism results confirmed that the decrease in the α-helical content of HSA is more on interaction with 16-6-16 MEA as compared to 16 MEA. In addition, the esterase activity of HSA was also done, which was found to decrease in presence of both 16 MEA and 16-6-16 MEA. Additionally, we also utilized computational approaches for deep insight into the binding of 16 MEA/16-6-16 MEA with HSA and the results well matched with our experimental results.