Ocular Fluid As a Replacement for Serum in Cell Cryopreservation Media

Varma, Vivek Phani; Devi, Lalitha; Venna, Naresh Kumar; Murthy, Ch. Lakshmi N.; Idris, Mohammed M.; Goel, Sandeep

doi:10.1371/journal.pone.0131291

Cited by 18 publications

(9 citation statements)

References 80 publications

(80 reference statements)

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“…To provide a source of cholesterol to the apoE, we added 10% fetal bovine serum (FBS), a common source of cholesterol ~310 μg/mL. Importantly, apoE is not present in FBS 48 allowing us to carefully control cholesterol loading. Cells were treated acutely (1 hr.)…”

Section: Resultsmentioning

confidence: 99%

The role of high cholesterol in age-related COVID19 lethality

Wang

Yuan

Pavel

et al. 2020

Preprint

132

177

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Coronavirus disease 2019 (COVID19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originating in Wuhan China in 2019. The disease in notably severe in elderly and those with underlying chronic conditions. The molecular mechanism as to why the elderly are vulnerable and why children are resistant is largely unknown. Understanding these differences is critical for safeguarding the vulnerable and guiding effective policy and treatments. Here we show loading cells with cholesterol from blood serum using the cholesterol transport protein apolipoprotein E (apoE) enhances the endocytic entry of pseudotyped SARS-CoV-2. Super resolution imaging of the SARS-CoV-2 entry point with high cholesterol showed markedly increased apparent diameter (~10% to 100 nm) and almost twice the total number of viral entry points. The cholesterol concomitantly traffics angiotensinogen converting enzyme (ACE2) to the viral entry site where SARS-CoV-2 docks to properly exploit entry into the cell. Furthermore, we show cholesterol enhances binding of SARS-CoV-2 to the cell surface which increases association with the endocytic pathway. Decreasing cellular cholesterol has the opposite effect. Based on these findings and known loading of cholesterol into peripheral tissue during aging and inflammation, we build a cholesterol dependent model for COVID19 lethality in elderly and the chronically ill. As cholesterol increases with age and inflammation (e.g. smoking and diabetes), the cell surface is coated with viral entry points and optimally assembled viral entry proteins. Importantly our model suggests high levels of cholesterol is most alarming in the tissue, not the blood. In fact, rapidly dropping cholesterol in the blood may indicate severe loading of cholesterol in peripheral tissue and a dangerous situation for escalated SARS-CoV-2 infectivity. Polyunsaturated fatty acids (PUFAs) oppose the effects of cholesterol and provide a molecular basis for eating healthy diets to avoid severe cases of COVID19.

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Section: Resultsmentioning

confidence: 99%

The role of high cholesterol in age-related COVID19 lethality

Wang

Yuan

Pavel

et al. 2020

Preprint

132

177

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“…In addition, reports also suggest that instead of FBS, buffalo ocular fluid can be used for the cryopreservation of mouse embryonic stem cells, and some primary cells such as mouse embryonic fibroblast cells, human peripheral blood mononuclear cells, and mouse bone marrow cells [ 36 ]. Moreover, it is also feasible to use instead of FBS for immature rodent and buffalo testis germplasm cryopreservation [ 37 ].…”

Section: Efforts Made To Reduce or Replace Fbsmentioning

confidence: 99%

Alternative to FBS in animal cell culture - An overview and future perspective

Chelladurai

Christyraj

Rajagopalan

et al. 2021

Heliyon

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Fetal bovine serum (FBS) is a widely used growth supplement in the in vitro culturing of animal and human cells, tissues and organs, notably due to the occurrence of abundant micro-and macronutrients, along with growth factors. Over the years, increasing demand, high price, batch-to-batch variability in quality and composition, increasing ethical concerns lead to the search for an alternative to FBS. Several approaches have been suggested and employed in the past, but none is implemented as widely as FBS, and each supplement has its own disadvantages. In this review, we described the importance of FBS in cell culture, discussed the issues associated with FBS use and presented the efforts made in the recent past to reduce or replace FBS. The potential of four different alternative sources to FBS, namely, bovine ocular fluid, sericin protein, human platelet lysate and earthworm heat inactivated coelomic fluid was evaluated. In the end, we present the conceptual perspective using the Human Platelet Lysate (HPL) and earthworm Heat Inactivated Coelomic Fluid (HI-CF) combination to alternate FBS and its context in scientific and economic impacts.

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“…Therefore, in this study, we focused on the development of an efficient vitrification method for tissues, which are a potential source of stem cells, using a cocktail of cryoprotectants. Animal serum proteins and albumin increase the efficiency of vitrification and reduce cryoinjury [18]. However, they can be contaminated by infectious agents and change the characteristics of cells [19].…”

Section: Introductionmentioning

confidence: 99%

Vitrification of Dog Skin Tissue as a Source of Mesenchymal Stem Cells

Son¹,

Jeong²,

Lee

et al. 2021

BioMed Research International

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The purpose of this study was to develop an efficient vitrification system for cryopreservation of dog skin tissues as a source of stable autologous stem cells. In this study, we performed vitrification using four different cryoprotectants, namely, ethylene glycol (EG), dimethyl-sulfoxide (Me2SO), EG plus Me2SO, and EG plus Me2SO plus sucrose, and analyzed the behaviors of cells established from warmed tissues. Tissues vitrified with 15% EG, 15% Me2SO, and 0.5 M sucrose had a normal histological appearance and the highest cell viability after cell isolation, and thus, this cocktail of cryoprotectants was used in subsequent experiments. We evaluated proliferation and apoptosis of cells derived from fresh and vitrified tissues. These cells had a normal spindle-like morphology after homogenization through subculture. Dog dermal skin stem cells (dDSSCs) derived from fresh and vitrified tissues had similar proliferation capacities, and similar percentages of these cells were positive for mesenchymal stem cell markers at passage 3. The percentage of apoptotic cell did not differ between dDSSCs derived from fresh and vitrified tissues. Real-time PCR analysis revealed that dDSSCs at passage 3 derived from fresh and vitrified tissues had similar expression levels of pluripotency (OCT4, SOX2, and NANOG), proapoptotic (BAX), and antiapoptotic (BCL2 and BIRC5) genes. Both types of dDSSCs successfully differentiated into the mesenchymal lineage (adipocytes and osteocytes) under specific conditions, and their differentiation potentials did not significantly differ. Furthermore, the mitochondrial membrane potential of dDSSCs derived from vitrified tissues was comparable with that of dDSSCs derived from fresh tissues. We conclude that vitrification of dog skin tissues using cocktail solution in combination of 15% EG, 15% Me2SO, and 0.5 M sucrose allows efficient banking of these tissues for regenerative stem cell therapy and conservation of genetic resources.

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Ocular Fluid As a Replacement for Serum in Cell Cryopreservation Media

Cited by 18 publications

References 80 publications

The role of high cholesterol in age-related COVID19 lethality

The role of high cholesterol in age-related COVID19 lethality

Alternative to FBS in animal cell culture - An overview and future perspective

Vitrification of Dog Skin Tissue as a Source of Mesenchymal Stem Cells

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