Hemoglobin Extracted From Perinereis aibuhitensis Acts as a Cell Culture Medium Supplement to Reduce Apoptosis

The environmental oxygen concentration is a crucial factor affecting cell proliferation. Owing to the reversible binding property of hemoglobin to oxygen, it can be utilized to regulate the oxygen concentration in vitro, and its ability to reduce apoptosis can be evaluated. In this study, a process comprising isolation, purification, and extraction was used to obtain hemoglobin from Perinereis aibuhitensis, a polychaete invertebrate. Extracts were separated and characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extract component identity was confirmed by matrix-assisted laser desorption time-of-flight mass spectrometry analysis, with the molecular weight determined as 412,216.6875 Da. The oxygen carrying capacity of P. aibuhitensis hemoglobin was comparable with that of human hemoglobin. P. aibuhitensis hemoglobin remarkably downregulated the apoptosis rate. Reactive oxygen species (ROS) assays confirmed the reduction in ROS production, enabling a better elucidation of the mechanism underlying the decrease in apoptosis. These results suggested that P. aibuhitensis hemoglobin is a natural oxygen carrier, that, owing to its low-cost and accessibility, can be considered a candidate for culture medium supplement to reduce the apoptosis rate.

Potential Effect of Human Platelet Lysate on in vitro Expansion of Human Corneal Endothelial Cells Compared with Y-27632 ROCK Inhibitor

Purpose: Corneal endothelial cell (CEC) therapy can be used as a promising therapeutic option for patients with various corneal endothelial dysfunctions. In this study, we compared the proliferative effect of human platelet lysate (HPL), as a xeno-free medium supplement, with Y-27632 Rho/rho-associated protein kinase (ROCK) inhibitor, as a wellknown proliferative and adhesive agent for CECs, and fetal bovine serum (FBS) as the control, in the culture medium of human corneal endothelial cells (HCECs). Methods: We isolated HCECs from human donors and treated the cells as three different treatment groups including 20% HPL only, 10 μM Y-27632 ROCK inhibitor, combination of 20% HPL and 10 μM Y-27632 ROCK inhibitor, and 20% FBS as the control group. ELISA cell proliferation assay and cell counting was performed on the treated cells. Finally, HCECs were characterized by morphology and immunocytochemistry (ICC). Results: There was no significant proliferative effect of HPL on cell proliferation compared with the cells treated with Y-27632 ROCK inhibitor or the combination of HPL and Y-27632 ROCK inhibitor, but all the respected treatments had significant inducible effect on cell proliferation as compared with FBS-treated cells. The cells grown in all three treatment groups exhibited CEC morphology. Also, there was a higher expression of Na+/K+-ATPase and ZO-1, as CEC characteristic markers, in the culture of HCECs treated with HPL as compared with FBS. Conclusion: HPL offers a xeno−free and affordable medium supplement for CEC expansion that can be used in clinical applications.

P–102 GM-CSF (granulocyte-macrophage colony-stimulating factor) as a sperm medium supplement improves sperm quality in Oligoastenoteratospermia (OAT) men by activating the PI3K/Akt pathway

Study question Can GM-CSF as a sperm medium supplement improve sperm quality in OAT men? Summary answer GM-CSF can be used to improve sperm quality in OAT men by activating the PI3K/Akt pathway. What is known already OAT patients have very low sperm parameters, including morphology, count, and motility, which can adversely affect the results of assisted reproduction technologies. It seems the development of sperm media is necessary to improve the sperm parameters of these patients. GM-CSF is a natural growth factor produced by the reproductive organs, culture media supplemented with GM-CSF is widely commercially available and enhances the embryo development and implantation rate. Studies show that this growth factor in the semen of infertile men is lower than that of fertile men. However, there is no study to assess the effect of GM-CSF on sperm quality. Study design, size, duration In the present study, Semen specimens were collected from 20 OAT patients who have male infertility factors, according to WHO criteria. After the swim-up washing procedure, each of the samples is divided into two groups; experiment, and control. In the experimental group, samples are incubated with a medium containing 2 ng/ml GM-CSF for one hour, yet, in the control group, the sperms are incubated without GM-CSF for the same time. Participants/materials, setting, methods The sperm motility was examined with phase-contrast microscopy, Eosin-nigrosin staining method was used to assess sperm viability. The expression of sperm glucose transporters (GLUT 1, 3) was determined using immunofluorescent staining, the ratio of pAkt to total Akt was assessed by the Western blotting method. Image J software was used to quantify results; the data was analyzed by SPSS software. P-value<0.05 was considered statistically significant. Main results and the role of chance As compared to the control group, supplementation with GM-CSF improved sperm progressive motility, enhanced GLUT 1 and 3, and p-AKT/AKT expression (P < 0.05). There was no significant difference between the viability of the control and experimental groups. Limitations, reasons for caution This study needs further investigations, including Real-time PCR for the genes of this signaling pathway which is ongoing. Wider implications of the findings: We showed for the first time that GM-CSF can improve sperm quality by influencing motility and energy metabolism in spermatozoa which can be affected by increasing the phosphorylation of AKT. This growth factor could be an appropriate supplement in sperm media for OAT patients. Trial registration number IRCT20200519047508N1

Effect of Vitamin C on In Vitro Maturation of Iraqi She-Camel Oocytes

This study aimed to know the effect of vitamin C on in vitro oocytes maturation of Iraqi she-camel with different techniques collection. Several oocytes collection technique have been used: Three hundred ninety oocytes were collected from 84 ovaries from Afak slaughterhouse within half to one hour of slaughter animal and transport by cool box contain normal saline 0.9% (20-25C°) to laboratory of Al-Diwaniyah veterinary Hospital within 1-2 hours. After washing the ovaries with normal saline, each 28 ovaries: oocytes collected by one of the following techniques: Aspiration, slicing, and dissection. Oocytes collected from the three techniques counted and graded. Only grades A and B were selected, and undergo in maturation process. and matured in maturation medium (M199-A) and incubated in CO2 incubator at 5% CO2, 38.5 Cº, and 90% humidity for 24h and supplement the media with 0, 25, 50, 100 µg/ml of vit.C for each technique. Aspirated oocytes were cultured the results was expended cumulus cells was 66.66% (66/99) and the appearance first polar body (F.P.B.) was 68.1% (45/66). Maturation medium supplement with 50µg/ml of vit. C higher rate (84% & 81.8 expended cumulus and F.P.B.) than other groups with significant (P<5%). Maturation oocytes by slicing technique were cultured, expended cumulus was 61.4% (59/96) and the appearance F.P.B. 62.7% (37/59). Maturation medium supplement with 50µg/ml of vit. C (79.16 & 73.68% expended cumulus and F.P.B.) higher rate than other groups with significant (P<5%). Maturation oocytes by dissection technique were cultured. expended cumulus was 57.2% (59/103) and the appearance F.P.B. 55.93% (33/59). Maturation medium supplement with 50µg/ml of vit. C higher rate than other groups with significant (P<5%).

Marine cyanobacterium Spirulina maxima as an alternate to the animal cell culture medium supplement

Serum is a stable medium supplement for in vitro cell culture. Live cells are used in stem cell research, drug toxicity and safety testing, disease diagnosis and prevention, and development of antibiotics, drugs, and vaccines. However, use of serum in culture involves concerns such as an ethical debate regarding the collection process, lack of standardized ingredients, and high cost. Herein, therefore, we evaluated the possibility of using edible cyanobacterium (Spirulina maxima), which is a nutrient-rich, sustainable, and ethically acceptable source, as a novel substitute for fetal bovine serum (FBS). H460 cells were cultured to the 10th generation by adding a mixture of spirulina animal cell culture solution (SACCS) and FBS to the culture medium. Cell morphology and viability, cell cycle, apoptosis, proteomes, and transcriptomes were assessed. We observed that SACCS had better growth-promoting capabilities than FBS. Cell proliferation was promoted even when FBS was replaced by 50–70% SACCS; there was no significant difference in cell shape or viability. There were only slight differences in the cell cycle, apoptosis, proteomes, and transcriptomes of the cells grown in presence of SACCS. Therefore, SACCS has the potential to be an effective, low-cost, and eco-friendly alternative to FBS in in vitro culture.

Human platelet lysate as a potential clinical-translatable supplement to support the neurotrophic properties of human adipose-derived stem cells

Background The autologous nerve graft, despite its donor site morbidity and unpredictable functional recovery, continues to be the gold standard in peripheral nerve repair. Rodent research studies have shown promising results with cell transplantation of human adipose-derived stem cells (hADSC) in a bioengineered conduit, as an alternative strategy for nerve regeneration. To achieve meaningful clinical translation, cell therapy must comply with biosafety. Cell extraction and expansion methods that use animal-derived products, including enzymatic adipose tissue dissociation and the use of fetal bovine serum (FBS) as a culture medium supplement, have the potential for transmission of zoonotic infectious and immunogenicity. Human-platelet-lysate (hPL) serum has been used in recent years in human cell expansion, showing reliability in clinical applications. Methods We investigated whether hADSC can be routinely isolated and cultured in a completely xenogeneic-free way (using hPL culture medium supplement and avoiding collagenase digestion) without altering their physiology and stem properties. Outcomes in terms of stem marker expression (CD105, CD90, CD73) and the osteocyte/adipocyte differentiation capacity were compared with classical collagenase digestion and FBS-supplemented hADSC expansion. Results We found no significant differences between the two examined extraction and culture protocols in terms of cluster differentiation (CD) marker expression and stem cell plasticity, while hADSC in hPL showed a significantly higher proliferation rate when compared with the usual FBS-added medium. Considering the important key growth factors (particularly brain-derived growth factor (BDNF)) present in hPL, we investigated a possible neurogenic commitment of hADSC when cultured with hPL. Interestingly, hADSC cultured in hPL showed a statistically higher secretion of neurotrophic factors BDNF, glial cell-derived growth factor (GDNF), and nerve-derived growth factor (NFG) than FBS-cultured cells. When cocultured in the presence of primary neurons, hADSC which had been grown under hPL supplementation, showed significantly enhanced neurotrophic properties. Conclusions The hPL-supplement medium could improve cell proliferation and neurotropism while maintaining stable cell properties, showing effectiveness in clinical translation and significant potential in peripheral nerve research.

Pseudo-outbreak ofBrevundimonas diminutaAttributed to Contamination of Culture Medium Supplement

We report an epidemiological investigation of a cluster ofBrevundimonas diminutaisolates cultured from sterile sites. Inoculation of supplement medium yielded growth ofB. diminuta. Molecular typing indicated likely contamination of the lot. NoB. diminutawas further isolated after replacement of the supplement with a new lot number.Infect Control Hosp Epidemiol2017;38:598–601