Article TitleAuthorPublishing DateKey WordsJournal TitleISSNOnline ISSNVolumeIssueLink to Full-Text Articles (PDF)AbstractDOILink to AbstractPublisherReference
1Low cost dipstick immunoblot assay development for bovine pregnancy diagnosisGirish K Srivastava2016.04Low cost, dipstick immunoblot assay, EIA kit bovine pregnancy diagnosis, diagonostic kit
Journal of Allbiosolution
2605-353511Low cost, affordable early pregnancy diagonsis kit is very deserving for livestock management and dairy business. This study proposes to develop a kit based on enzyme immunoassay (EIA). New Zealand white rabbits primed with 11-α-OH progesterone hemisuccinate-BSA conjugate were boosted with progesterone antigen for crude antisera production. Progesterone antibody was purified using an immuno affinity chromatography column packed with 6-amino hexanoic acid sepharose 4B beads conjugated with progesterone carboxy methyl oxime and coated on nitrocellulose paper dipsticks. The 11-α-OH progesterone hemisuccinate-penicillinase conjugate solution was purified using Sephadex G-50 size exclusion column. All conjugates were prepared using carbodiimide reaction. Optimum pH and temperature for penicillinase enzyme highest activity and iodine concentration for starch iodine substrate solution were determined. Checker board anlysis and titration were performed to optimize antibody, conjugate and milk dilutions for kit. Further kit was validated with randomly selected 20 cow milk samples for pregnancy diagnosis. Chromatography data showed peaks for fractions containing highly purified progesterone antibody and progesterone penicillinase conjugate respectively. Optimum dilutions of antibody, conjugate and milk samples were 1:20, 1:40, 1:3 (or 1:4) respectively to differentiate between 1 (non pregnant) and 5 (pregnant) ng/ml of progesterone concentration. Standard conditions of kit optimum function are pH 7.0 and 35 ºC. Penicillin solution was stable at 4 ºC for 3 weeks. Excellent correlation was observed for pregnancy diagnosed by developed kit to detect progesterone concentration in 20 milk samples and conventional rectal palpation method. Developed low cost dipstick EIA kit can be used for early pregnancy diagnosis successfully.

2Schwann cells culture with adipose stem cells and neuregulin to determine influencing factors applicable in nerve regenerationMaría Nelcy Orozco García1; Manuel J. Gayoso; Sara Gayoso del Villar; Girish K. Srivastava; Manuel Garrosa2016.11Nerve Regeneration, Schwann, Adipose-derived mesenchymal stem cells, Neuregulin, Co-cultureJournal of Allbiosolution2605-353511Schwann cell (SC) cultures are required for neural regeneration, as for example in injured nerves. However, SC culture procedures need improvements. This study evaluates and compares growth of SC cultures without and with neuregulin, and in co-cultures with adipose mesenchymal stem cells (ASCs). Rat originated SCs and ASCs were cultured, maintained and subcultured to prepare monocultures and co-cultures for this study. Morphological, immunohistochemical and quantitative evaluations were performed at 48 and 96 h. In monocultures SCs were S100 and vimentin positive, appeared bipolar with small somata containing small and elongated nuclei, and tended to contact on their ends forming chains or clustering around other cell types. ASCs were CD 105 and vimentin positive, showed fibroblast-like shape with large rounded nuclei and distinct nucleoli, became larger along time and contacted with the neighboring cells. In SC-ASC co-culture, a few ASCs appeared in triangular shape, their CD105 positivity turned weaker and became vimentin negative at 4 days confirming ASC differentiation initiation towards SC. Fibroblasts appeared in a small proportion in each culture. Cell counting and further ANOVA analysis revealed significant differences for cell growth between 48 and 96 h in each culture, whereas across different cultures, significant differences were found only between SC cultures grown without neuregulin and rest of SC cultures. In co-cultures ASC proliferated faster. Neuregulin increases SC cultures growth about 4 times faster than SC cultures alone. A similar SC proliferation increase was observed in co-cultures of SC with ASC at 96 h, suggesting possible role of ASC released growth factors in SC growth.
3Phenol as a positive control in cytotoxicity test methodsGirish K Srivastava; José Carlos Pastor 2018.04Phenol, cytotoxicity, ISOJournal of Allbiosolution2605-353511Cytotoxicity test is a primary indictor of biocompatibility and safety of medical
devices. The ISO 10993 has recommended several in vitro cytototoxicity test
methods and parameters. One of them is possibility to apply phenol as a
positive control. Phenol is cytotoxic to cell and tissue cultures over determined
concentrations. The paper describes errors generated in experimental results if
phenol is used for long period in cytototoxic test methods. The ARPE-19 and
L929 cell cultures are prepared in 96 wells plate following ISO 10993
recommendations and exposed to positive, negative and test samples. Different
concentrations of phenol (0.025, 0.1, 0.2. 0.4, 1.6 mg/ml as well as 1x (1.6
mg/ml), 3x, 66.6% of 1x and 3x) are prepared with culture medium and used in
different experimental plans. The cell cultures were exposed, following direct
contact cytotoxicity method, directly to phenol concentration for 30 and 60
minutes and then grown for 24 and 72 hours, or were, following extract contact
cytotoxicity method, incubated 72 hours with phenol concentrations. The cell
culture viability was measured using the MTT and XTT assay. The data are
analyzed statistically and errors generated due to phenol samples were
detected. The results showed the cell culture had lost their viability (<1%) at 1.6
mg / ml concentration of phenol on 30 minutes exposure. On short period
exposure (up to 60 minutes in this study), there were no effects on viability of
cell cultures of neighboring wells of a culture plate. However, where cell cultures
were incubated for a long period, 72 hours, with phenol, the cell culture of
neighboring wells had also lost their viability. This lost decreases as far as wells
are from the wells that contained phenol. The results showed the L9292 cell
cultures were more sensitive to phenol than ARPE-19 cell cultures. The study
concludes that including phenol presence as a positive control for long period in
an experimental plan can generate errors in data; which concludes into false
positive for a test sample.
4Direct contact method with new technical steps essential to detect cytotoxicity of a volatile substance. Medical device quality assurance.Girish K Srivastava; Maria T Garcia; José Carlos Pastor2017.12Medical device quality assurance, Direct contact method, Cytotoxicity, Volatile substancesJournal of Allbiosolution2605-353511Medical devices need to pass ISO recommended safety assessments
before clinical use. Perfluorocarbon liquids (PFOs) are used in intraocular
surgery. Recent cases of blindness raised questions against current
cytotoxicity test methods applied for safety assessments of PFOs. The
study proposes to assess the cell culture responses to different PFOs by
applying a direct contact method incorporating few new technical steps for
testing volatile substances such as PFOs. Limitations of current methods;
extract dilution exposure and agarose overlay methods, were recorded.
ARPE-19 cell cultures were prepared, and the direct contact and extract
dilution exposure methods were performed for different PFOs. Agarose
overlay method was performed partially. Results showed that for non toxic
PFOs, the ARPE-19 cell cultures showed different tendencies of growths at
24 and 72 hours for PFOs of different manufacturers maintaining always
cell viability ≥ 70%; however, the difference was not significant for PFOs of
a same manufacturer. The toxic PFOs were detected toxic which extract
dilution exposure method failed to detect. There were several limitations
detected in applying the current cytotoxicity test methods. One crucial
limitation was that it was unknown if a quantity of PFO or toxicity
components of PFO reached in contact with cell culture, which was not in
the case of new direct contact method. It has detected non-toxic and toxic
PFOs lots which were manufactured differently. The study strengthens the
arguments that a direct cytotoxicity method with the new technical steps is
essential for safety assessments of volatile substances and guarantying
safety of such medical devices.
5YIGSR sequence contained elastin like recombinamer for sustaining retinal pigment epithelium as a possible tool for retinal regenerative medicineGirish K. Srivastava; Laura Martin; David Rodríguez-Crespo; Amar K. Singh; Manuel J. Gayoso; José C. Rodríguez-Cabello; José C. Pastor2018.06ELR; YIGSR; RPE; AMD; BiomaterialJournal of Allbiosolution2605-353511Retinal pigment epithelial (RPE) cell replacement therapy is promising for AMD
patients. However, it needs a suitable surface to sustain RPE characteristics.
Constructed elastin like recombinamer containing YIGSR sequence (ELR-
YIGSR), using solvent-casting onto glass cover slips followed by cross-linking,
was evaluated along with TCP polystyrene (control) for ARPE19 cells
sustainability (cell adhesion, viability, proliferation, epithelial characteristics and
cellular junctions) at 4, 24, 72 and 120 hours. Cell viability was over 90% at 72
and 120 hours. Numbers of cells adhered (42.29±24.24%) at 4 hours and grew
at 4, 24, 72, and 120 hours on ELR-YIGSR surface was significantly lower
(25±16, 63.5±25, 420±60 and 662±45) than that of control TCP polystyrene
(49.5±25.21, 190.5±57.27, 768±67.81 and 810.3±145.2). Microscopic results

confirmed epithelial characteristics (hexagonal morphology, Ecad and ZO-
1protein expressions, and tight and gap junctions). Thus, it confirmed ELR-
YIGSR surface sustained RPE cell characteristics. However, this needs

subsequent studies with fresh human RPE cells to conclude its application in
6Quantification of pigment epithelium-derived factor (PEDF) in an ex vivo coculture of retinal pigment epithelium cells and neuroretinaSalvatore Di Lauro; Maria Teresa Garcia-Gutierrez; Ivan Fernandez-Bueno2019.11Ex vivo model; Retinal pigment epithelium (RPE); Neuroretina; Pigment Epithelium-Derived Factor (PEDF); NeuroprotectionJournal of Allbiosolution2605-353521To quantify the pigment epithelium-derived factor (PEDF) levels in a coculture
model of physically separated neuroretina and retinal pigment epithelium (RPE) cells, and to point out its potential role in neuroretinal maintenance.RPE cells and neuroretina explants were isolated from porcine eyes. RPE cells were expanded and seeded on the bottom of Transwell® culture inserts. Neuroretina explants were cultured alone (controls) on Transwell® culture membranes or supplemented with RPE cells in the same wells but physically separated by the culture membrane, during 9 days. PEDF concentration in the culture medium at 3, 5, 7 and 9 days of culture was determined by enzyme-linked immunosorbent assays (ELISA) specific for porcine samples. Mean statistical analysis were performed with Pair-wise Student’s-tests. Culture medium collected from neuroretina cultures without and with RPE cells contained detectable levels of PEDF in both conditions and at all evaluated time-points. At
3, 5, and 7 days and through the whole time of culture PEDF concentration was
significantly higher in cocultures with RPE cells (p<0.05). RPE cells neuroprotective role may be linked to the beneficial effects of neurotrophic factors, such as PEDF, secreted or induced by RPE cells during co-culture.
7Effect of cell growth supporting surface on phagocytosisGirish K Srivastava; David Rodriguez-Crespo; J. Carlos Pastor 2018.09Retinal pigment epithelial cells, phagocytosis, cell growth supporting surface, latex beads Journal of Allbiosolution2605-353521Phagocytosis is involved in several functions including nutrient uptake,
immune response, inflammation, tissue homeostasis, cellular apoptotic
bodies and debris elimination. Its dysfunction has impacts on normal
functioning of an organism. It is involved in several diseases including the
AMD pathogenesis, and treatment approaches. Mostly an initial proof of
concept is tested in a laboratory conditions. Cell and tissue cultures need
proper surfaces to grow and maintain them in laboratory conditions. The
study aims to evaluate effect of two surfaces; tissue culture plate polystyrene
and glass coverslip, on phagocytosis performed by RPE cells growing on
each surface. Fresh RPE cells and ARPE-19 cell line were grown in
corresponding cell culture conditions. Phagocytosis in fresh RPE cell cultures
was detected and recorded. ARPE-19 cells were incubated for 4 and 24
hours with 0.2 and 1 μl red fluorescent latex beads, and then images were
taken to measure the average numbers of cells, cells performing no
phagocytosis and phagocytosis and engulfed red fluorescent latex beads.
Results showed that RPE cell membrane was forming blebs releasing black
dots like particles as cell debris in cell culture medium. Subsequently
cytoplasmic membrane extensions were formed which had engulfed black
dots like particles. ARPE-19 cells are a good cellular model for performing
initial in vitro test in comparison to fresh RPE cells. There was no significant
difference between numbers of ARPE-19 cells growing on polystyrene and
glass surface performing latex bead phagocytosis. Both had engulfed red
fluorescent latex beads; however, numbers of engulfed beads was
significantly higher for glass surface than polystyrene surface. Results
support the hypothesis that surface of cell culture plates affects the cellular
phagocytosis mechanism, which play a critical role in the functions in many
cases such as in vivo retina. These findings are very crucial when it comes to
transfer a treatment approach based on phagocytosis to the clinics and that
the materials and the conditions of the experiments can have very important
8Dimethyl sulfoxide toxic and stimulation effects in retinal pigment epithelial cellsGirish K. Srivastava; Antonio Dueñas-Laita; J Carlos Pastor2019.08Cell toxicity, cell stimulation, Dimethyl sulfoxide, DMSO, RPE cellsJournal of Allbiosolution2605-353521Dimethyl sulfoxide (DMSO) is used for drug formulations as a active pharmaceutical ingredients (API) or
vehicle of an API. Eye is considered as a relatively isolated organ, protected by barriers similar to the
blood-brain one. Thus many systemically administered drugs are not able to reach adequate intraocular
levels because they are unable to cross these blood-ocular barriers. Direct injection of drugs into the
vitreous cavity has become very popular in recent years. Therefore, it is crucial to establish the level of
toxicity and safety of the drugs, since the intraocular tissues are especially sensitive to certain situations
that would be perfectly tolerated by other routes of administration. This study has been focused on
evaluating the effect on RPE cells directly exposed to DMSO for short to long periods by analyzing cell
morphology, cell culture ́s confluence level, cell proliferation rate and viability. ARPE-19 cell cultures
were exposed to 10%, 5%, 2%, 1%, 0.50%, 0.20% and 0.10% DMSO concentrations and grown for 1, 4
and 14 days. Microscopic observations were performed at 3 hours and 4 days to note any changes in cell
morphology and cell culture confluence level. MTT assay was performed to assess cell viability / toxicity
and proliferation. The results showed that cells exposed to 10% to 0.50% DMSO were affected for cell
morphology and viability / proliferation or only morphology or level of cell culture confluence, even
showing dead cell debris as they grew from 1 to 14 days. However, they retained cell morphology,
viability and confluent level at concentrations of 0.20% to 0.01% DMSO. But even at lower
concentrations, 0.05% to 0.01%, it was noted significant cell proliferations. This study provides data for
DMSO effect assessments in RPE cell culture in in vitro conditions for measuring cell morphology,
viability / toxicity and proliferation, which could be useful in drug formulations. However, this needs to
be evaluated in clinical practice as well.
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