Rodent Bone Marrow Research Articles

Quinoline is more genotoxic than 4-methylquinoline in hiHeps cells and rodent liver.

Environmental pollutants, such as quinoline (QN) and 4-methylquinoline (4-MeQ), may be genotoxic and carcinogenic. Earlier studies, including in vitro genotoxicity tests, indicated that 4-MeQ is more mutagenic than QN. However, we hypothesized that the methyl group of 4-MeQ favors detoxication over bioactivation, and this factor may be overlooked in in vitro tests that do not incorporate supplementation with cofactors for enzymes that catalyze conjugation reactions. We used human induced hepatocyte cells (hiHeps), which express such enzymes, and compared the genotoxicity of 4-MeQ and QN. We also carried out an in vivo micronucleus (MN) test in rat liver, since 4-MeQ is not genotoxic in rodent bone marrow. In the Ames test and the Tk gene mutation assay, with rat S9 activation, 4-MeQ was more mutagenic than QN. However, QN induced significantly higher MN frequencies in hiHeps and rat liver than did 4-MeQ. Furthermore, QN upregulated genotoxicity marker genes much more than did 4-MeQ. We also investigated the roles of two important detoxication enzymes, UDP-glucuronosyltransferases (UGTs) and cytosolic sulfotransferases (SULTs). When hiHeps were preincubated with hesperetin (UGT inhibitor) and 2,6-dichloro-4-nitrophenol (SULT inhibitor), MN frequencies were elevated approximately 1.5-fold for 4-MeQ, whereas no significant effects were seen for QN. This study shows that QN is more genotoxic than 4-MeQ, when the roles of SULTs and UGTs in detoxication are considered and our results may improve understanding the structure-activity relationships of quinoline derivatives.

Genotoxicity test battery – An assessment of its utility in early drug development

Formal requirements for genotoxicity testing of drug candidates to support clinical entry have been in place since the issue of initial regulatory guidance over 25 years ago and subsequent update a decade ago. An evaluation of such testing, supporting first clinical entry of 108 small molecule drug candidates over the last decade, showed that the most common approach (75 % of tested compounds) was for a Good Laboratory Practice test battery in the form of 2 in vitro (a bacterial reverse mutation and a mammalian cell) assays and one in vivo assay. The majority of other tested compounds involved in vitro testing only in bacterial reverse mutation and mammalian cell assays. Testing using a bacterial reverse mutation assay and an in vivo assessment of genotoxicity with 2 different tissues was limited to 2 occasions. For in vitro mammalian cell testing, the chromosome aberration test was most commonly used (70 % occasions), followed by a micronucleus test (16 % occasions) or a mouse lymphoma assay (14 % occasions). For in vivo evaluation, the most common test was a rodent bone marrow micronucleus test (87 % occasions). A positive in vitro mammalian cell assay result was seen on 13 % occasions but was not confirmed with further in vivo testing and the drug candidates were taken into the clinic. In conclusion, the present evaluation showed that the current test battery paradigm for genotoxicity testing has an integral part in supporting clinical entry to confirm candidate drugs taken into the clinic are unlikely to have genotoxic activity.

A Novel Enzymatic Digestion Approach for Isolation and Culture of Rodent Bone Marrow Mesenchymal Progenitors.

Bone marrow mesenchymal stem cells (MSCs) are promising therapeutic tools for tissue repair and treatment of a number of human diseases. As a result, there is substantial interest in characterizing and expanding these cells to uncover their therapeutic potential. Bone marrow mesenchymal progenitors, containing both MSCs and their proliferative progeny, are commonly isolated from the central region of rodent long bones. However, challenges exist in expanding these central mesenchymal progenitors in culture. We have designed an enzymatic digestion protocol to isolate mesenchymal progenitors within rodent long bones that resides close to the bone surface, which we termed endosteal mesenchymal progenitors. These cells are more metabolically active and more responsive to external stimuli compared to central mesenchymal progenitors. Therefore, they represent a biologically important target for MSC research. This chapter describes the approach in detail how to isolate and culture endosteal mesenchymal progenitors as well as their central counterparts from rodent long bones.

Biocompatibility of Bone Marrow-Derived Mesenchymal Stem Cells in the Rat Inner Ear following Trans-Tympanic Administration.

Recent advancements in stem cell therapy have led to an increased interest within the auditory community in exploring the potential of mesenchymal stem cells (MSCs) in the treatment of inner ear disorders. However, the biocompatibility of MSCs with the inner ear, especially when delivered non-surgically and in the immunocompetent cochlea, is not completely understood. In this study, we determined the effect of intratympanic administration of rodent bone marrow MSCs (BM-MSCs) on the inner ear in an immunocompetent rat model. The administration of MSCs did not lead to the generation of any oxidative stress in the rat inner ear. There was no significant production of proinflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-12, due to BM-MSCs administration into the rat cochlea. BM-MSCs do not activate caspase 3 pathway, which plays a central role in sensory cell damage. Additionally, transferase dUTP nick end labeling (TUNEL) staining determined that there was no significant cell death associated with the administration of BM-MSCs. The results of the present study suggest that trans-tympanic administration of BM-MSCs does not result in oxidative stress or inflammatory response in the immunocompetent rat cochlea.

Evaluation of genotoxic effects of methanolic extract of brown seaweed Stoechospermum marginatum

Seaweeds are considered as a valuable source of bioactive compounds produced as secondary metabolites having a broad spectrum of biological activities. Recently seaweed has been extensively utilized by pharmaceutical, chemical and nutraceutical industries as valuable sources of raw material of natural origin. The present study aimed at evaluating the mutagenic and genotoxic effects of methanolic extract of brown seaweed Stoechospermum marginatum. The test extract was assessed for its potential mutagenic effect using in vitro bacteria reverse mutation assay (Ames test) and for its potential clastogenic and DNA damaging effects using in vivo rodent bone marrow micronucleus and alkaline comet assay respectively. The test extract did not produce a two-fold increase or a reproducible dose-dependent increase in the number of revertant colonies in all the bacterial tester strains at the highest dose of 5000 µg/plate tested both with and without metabolic activation. No significant clastogenic and DNA damaging effects were observed at the highest dose of 2000 mg/kg body weight when the test extract was assayed in-vivo in mice bone marrow erythrocytes and peripherally in lymphocytes respectively. The result obtained from this study revealed that methanolic extract of Stoechospermum marginatum is safe to living cells genome and does not produce significant mutagenic and genotoxic effect when assayed in both in vitro and in vivo testing systems.

Effect of Bone Marrow-Derived Mesenchymal Stem Cells on Cochlear Function in an Experimental Rat Model.

Mesenchymal stem cell (MSC) therapy is an emerging treatment modality for various human diseases. Although induced pluripotent stem cells have been explored for the restoration of hearing, the potential of MSCs as a therapeutic strategy for various cochlear insults is not precisely known. MSCs possess anti-inflammatory, anti-apoptotic and neuroprotective properties, making them an attractive target for the treatment of inner ear disorders such as hair cell damage in response to inflammation. Most of the previous studies have used immunosuppression or the complex surgical techniques to deliver stem cells into the cochlea. However, no information is available regarding the biocompatibility and safety of MSCs in the inner ear in immunocompetent cochlea. The aim of the present study was to determine the effect of non-surgical administration of rodent bone marrow derived MSCs (BM-MSCs) through transtympanic delivery on the cochlear function and to assess any adverse effects on the auditory system employing a rat model without immunosuppression. We observed that the transtympanic administration of BM-MSCs has no significant effect on the hearing thresholds as determined by auditory brainstem response and distortion product otoacoustic emissions. Histopathological examination revealed no recruitment of inflammatory leukocytes and edema in the cochlea of BM-MSCs administrated rats. The results of this study suggest that transtympanic administration of BM-MSCs is safe and can be explored in providing otoprotection against cochlear insults. Anat Rec, 303:487-493, 2020. © 2019 American Association for Anatomy.

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OBJECTIVES/SPECIFIC AIMS: To assess the efficacy of exogenous administration of MSCs in animal models of HIE. METHODS/STUDY POPULATION: Adhering to the Systematic Review Protocol for Animal Intervention Studies, a systematic search of English articles was performed using MEDLINE, Web of Science, CINAHL, and Google Scholar. Search term items included mesenchymal stem/stromal cell, hypoxic ischemic encephalopathy, asphyxia, cerebral ischemia, and neonatology. We selected randomized and nonrandomized studies that examined in vivo neonatal models of induced HIE. We excluded studies that combined MSCs with an adjunct therapy. Data were collected on study specifics, MSC characteristics, and outcome measurements. The primary outcome was efficacy of MSC treatment, assessed by functional neurologic measures (cognitive, motor, sensory). Risk of bias was assessed using the SYRCLE’s risk of bias tool and we used the ARRIVE guidelines to describe the quality of study reporting. RESULTS/ANTICIPATED RESULTS: A total of 17 preclinical publications focusing on MSC therapy for HIE met our inclusion criteria. Fifteen of the studies (88%) induced HIE in rodents by ligating the common carotid artery followed by a period of hypoxic exposure. Nine (53%) studies derived their MSCs from rodent bone marrow, whereas the other investigators provided xenografts from human bone marrow or umbilical cord-derived MSCs. Range of MSC dose was between 0.25 and 3.5× 106 cells with 71% of the experiments transplanting the MSCs intranasally or intracerebral. Three studies (18%) administered multiple doses. The cylinder rearing test was the most common (73%) sensorimotor functional outcome performed in the first month following the establishment of HIE. All studies demonstrated a reduction in asymmetrical paw preference after receiving MSC therapy. Lesional size was assessed, using neuroimaging or histologic evaluations, and the majority of studies showed a decreased insult following MSC therapy. DISCUSSION/SIGNIFICANCE OF IMPACT: MSC treatment demonstrates improved functional and structural outcomes that are encouraging for future translational studies.

Unacylated ghrelin promotes adipogenesis in rodent bone marrow via ghrelin O-acyl transferase and GHS-R1a activity: evidence for target cell-induced acylation

Despite being unable to activate the cognate ghrelin receptor (GHS-R), unacylated ghrelin (UAG) possesses a unique activity spectrum that includes promoting bone marrow adipogenesis. Since a receptor mediating this action has not been identified, we re-appraised the potential interaction of UAG with GHS-R in the regulation of bone marrow adiposity. Surprisingly, the adipogenic effects of intra-bone marrow (ibm)-infused acylated ghrelin (AG) and UAG were abolished in male GHS-R-null mice. Gas chromatography showed that isolated tibial marrow adipocytes contain the medium-chain fatty acids utilised in the acylation of UAG, including octanoic acid. Additionally, immunohistochemistry and immunogold electron microscopy revealed that tibial marrow adipocytes show prominent expression of the UAG-activating enzyme ghrelin O-acyl transferase (GOAT), which is located in the membranes of lipid trafficking vesicles and in the plasma membrane. Finally, the adipogenic effect of ibm-infused UAG was completely abolished in GOAT-KO mice. Thus, the adipogenic action of exogenous UAG in tibial marrow is dependent upon acylation by GOAT and activation of GHS-R. This suggests that UAG is subject to target cell-mediated activation – a novel mechanism for manipulating hormone activity.

“Brazilian ginseng” (Pfaffia glomerata Spreng. Pedersen, Amaranthaceae) methanolic extract: cytogenotoxicity in animal and plant assays

Pfaffia glomerata (Spreng) Pedersen is a medicinal plant largely used as an adaptogenic herb. The species is naturally found in Africa and Americas and is the subject of pharmaceutical and commercial interest, mainly due to the accumulation of β-ecdysone in its roots. Brazil is considered the greatest supplier of P. glomerata in the world. Due to the morphological similarity of its roots to those of Panax ginseng (Korean ginseng), the species became known as the Brazilian ginseng. In this study, we performed the cytogenotoxic evaluation of P. glomerata root extract in rodent bone marrow micronucleus and Allium cepa assays. Taking into consideration the therapeutic dosage of P. glomerata, three methanolic extract dilutions were used in the Micronucleus assay. Each animal was treated with two consecutive doses with a 24-h interval. To assess the cytogenotoxicity of the extract, 2000 polychromatic erythrocytes per animal were analyzed. For the Allium cepa assay, onion seeds were exposed during 24h to six methanolic extract dilutions. Cell cycle and chromosomal alterations were quantified. The results of the Micronucleus assay showed genotoxic effects for all treatments investigated, considering the increase in the number of micronucleated cells. In the A. cepa assay the methanolic extract induced alterations in the cell cycle. Aneugenic and clastogenic effects were observed. The results indicate cytogenotoxic activity of P. glomerata extract in animal and plant cells. Thus, the ingestion of P. glomerata for medicinal purposes should be done carefully. The results also lead to the possibility of investigating the potential of the species in the research of prospective compounds for cancer treatment and for isolation of biologically active substances.

The phytoecdysteroid β-ecdysone is genotoxic in Rodent Bone Marrow Micronuclei and Allium cepa L. Assays

Ethnaopharmacologial relevanceIn South America, the β-ecdysone ecdysteroid has been found in species of the genus Pfaffia Mart. Due to the similar morphology of its roots to the Panax ginseng C. A. Mey. (Korean ginseng), some species of this genus has been known as Brazilian ginseng and have been used as tonic and aphrodisiac, as well as for the treatment of diabetes and rheumatism. Aim of the studyHere we report a cytogenotoxic evaluation of β-ecdysone (a natural ecdysteroid found in plants) in Rodent Bone Marrow Micronuclei and Allium cepa Assays. Materials and methodsThree β-ecdysone (pure) concentrations (based in human therapeutic dosage) were used in the Micronucleus Assay. The animals were treated during two consecutive days. Micronucleated cells were counted in 2000 polychromatic erythrocytes per animal. For A. cepa L. Assay, one β-ecdysone concentration was analyzed. The onions bulbs were exposed for 24h. ResultsThe Micronucleus Assay showed genotoxic effects for all treatments, expressed by an increase of micronucleated cells. In A. cepa L. Assay, cell abnormalities associated to the malfunction/non-formation of mitotic spindle (aneugenic effect) and chromosomal bridges (clastogenic effect) were observed. ConclusionsThe results indicate a cytogenotoxic activity of β-ecdysone. Therefore, the popular use of Pfaffia and others species containing β-ecdysone should be considered with caution.

Bone marrow spontaneous lesions in rodents from nonclinical 104-week carcinogenicity studies

The authors performed a retrospective study to determine the incidences and range of spontaneous lesions in the bone marrow (sternum and femur) of control mice and rats. Data was collected from 2186 mice (Crl:CD-1(ICR)BR), and 2347 rats (Han Wistar and CD(SD) rats) from the control dose groups of 104-week carcinogenicity studies carried out between 2005 and 2014. The incidence of spontaneous lesions in the bone marrow was higher in mice than in rats, and in both species non-neoplastic lesions were more common than neoplastic lesions. In mice, the most common non-neoplastic lesions in the bone marrow were increased cellularity, pigmented macrophages, and decreased cellularity, and the most common neoplastic lesions were malignant lymphoma, granulocytic leukemia and histiocytic sarcoma. There were occasional sex and site differences (sternum marrow vs femur marrow) in the incidence of a few bone marrow lesions in mice. In rats, the most common non-neoplastic lesions were increased cellularity and stromal fibrosis, and the most common neoplastic lesion was malignant lymphoma. In rats, no sex predilection in the incidence of bone marrow lesions was apparent, and there were no significant site differences in the incidence of lesions. To the best knowledge of the authors, there are no recent reports on spontaneous pathological findings in bone marrow of rodents, and we believe that these results will facilitate the interpretation of background findings and/or their increased incidence in carcinogenicity studies.

Evaluation of 4-methylimidazole, in the Ames/Salmonella test using induced rodent liver and lung S9.

4-methylimidazole (4-MeI) is formed by the interaction of ammonia with reducing sugars and low levels have been identified as a by-product in coffee, soy sauce, wine, dark beers, soft drinks, and caramel colors. The 4-MeI has been reported to induce alveolar/bronchiolar tumors in mice but not rats. Its mechanism of action is unlikely to be due to genotoxicity as 4-MeI does not induce mutation in Salmonella typhimurium and does not induce micronuclei in rodent peripheral erythrocytes or bone marrow cells. However, the question of whether genetically reactive intermediates could be formed via lung-specific metabolism has not previously been addressed. The 4-MeI was tested for its ability to induce mutation in five standard Ames strains of S. typhimurium using induced rat (F344/N) and mouse (B6C3F1) liver and lung S9 as a source of exogenous metabolism. The chemicals were tested in an OECD 471-compliant bacterial reverse mutation assay, using both plate-incorporation and pre-incubation methodologies, together with 10% S-9 metabolic activation. No induction of mutation (as measured by an increase in revertant colonies) was observed and it was concluded that 4-MeI was not mutagenic in S. typhimurium using either rodent liver or lung S9 for exogenous metabolism.

Differential membrane marker expression in adult rodent bone marrow mesenchymal and neural crest stem cells

Differential membrane marker expression in adult rodent bone marrow mesenchymal and neural crest stem cells

Priming Wharton's jelly-derived mesenchymal stromal/stem cells with ROCK inhibitor improves recovery in an intracerebral hemorrhage model.

Mesenchymal stromal/stem cells (MSCs) have the potential to differentiate into neuron-like cells under specific conditions and to secrete paracrine factors for neuroprotection and regeneration. Previously, Rho-kinase inhibitors have been reported to potentiate differentiation of rodent bone marrow MSCs into neuron-like cells induced by CoCl2 (HIF-1α activation-mimicking agent). Here, a strategy of priming MSCs with fasudil, a Rho-kinase inhibitor, was investigated using Wharton's jelly-derived MSCs (WJ-MSCs) to improve recovery in a rat model of intracranial hemorrhage (ICH). In vitro culture of WJ-MSCs by co-treatment with fasudil (30 μM) and CoCl2 provoked morphological changes of WJ-MSCs into neuron-like cells and increased the expression of neuronal markers. Assessment of the secretion profiles showed that fasudil (30 μM) specifically increased glial cell line-derived neurotrophic factor (GDNF) among the secreted proteins at the transcription and secretion levels. For in vivo experiments, WJ-MSCs primed with fasudil (10 μM, exposure for 6 h) were transplanted into ICH rats with HIF-1α upregulation 1 week after injury, and neurological function was assessed via rotarod and limb placement tests for 7 weeks after transplantation. The group with WJ-MSCs primed with fasudil showed improved functional performance compared with the non-primed group. Accordingly, the primed group showed stronger expression of GDNF and higher levels of microtubule-associated protein 2 and neurofilament-H positive-grafted cells in the ICH lesion 3 weeks after transplantation compared with the non-primed group. Therefore, this work suggests that priming WJ-MSCs with fasudil is a possible application for enhanced cell therapy in stroke, with additional beneficial effect of up-regulation of GDNF.

Genetic toxicity of high-boiling petroleum substances

There are several specific types of high-boiling petroleum substances (HBPS) having final boiling points >343°C), in which genetic toxicity can be related to the content of polycyclic aromatic compounds (PACs), specifically crude oils, gas oils, heavy fuel oils, lubricant base oils, waxes and aromatic extracts. Evaluation of optimized Salmonella tests covering over 250 samples from 43 types of HBPS revealed that gene mutation can be determined for these substances using a protocol optimized for the detection of mutagenic PAC. The outcomes of modified Salmonella assays can be predicted using HBPS compositional information as input to a newly developed statistical model. The general outcome of the optimized Salmonella assay can be predicted for an untested substance based on its Aromatic Ring Class (ARC) profile. Review of the results from numerous cytogenetic tests showed that although a few positive study results have been reported, most HBPS do not produce chromosomal effects when tested in rodent bone marrow assays or in in vitro chromosomal aberration assays. Results of both bacterial and cytogenetic studies can be used to satisfy genetic toxicity endpoints for the HBPS category substances.

Identification of Free Nitric Oxide Radicals in Rat Bone Marrow: Implications for Progenitor Cell Mobilization in Hypertension

Nitric oxide (NO) has been implicated in matrix metallopeptidase 9 (MMP9)-dependent mobilization of hematopoietic stem and progenitor cells from bone marrow (BM). However, direct measurement of NO in the BM remained elusive due to its low in situ concentration and short lifetime. Using NO spin trapping and electron paramagnetic resonance (EPR) spectroscopy we give the first experimental confirmation of free NO radicals in rodent BM. NO production was quantified and attributed to enzymatic activity of NO synthases (NOS). Although endothelial NOS (eNOS) accounts for most (66%) of basal NO, we identified a significant contribution (23%) from inducible NOS (iNOS). Basal NO levels closely correlate with MMP9 bioavailability in BM of both hypertensive and control rats. Our observations support the hypothesis that inadequate mobilization of BM-derived stem and progenitor cells in hypertension results from impaired NOS/NO/MMP9 signalling in BM, a condition that may be corrected with pharmacological intervention.

Allogeneic Cytokine-Induced Killer Cell-Dependent Eimination of Host Dendritic Cells Leads to Less Graft-Versus-Host Disease.

AbstractAbstract 3011Cytokine-induced killer (CIK) cells are ex vivo-expanded T lymphocytes expressing both natural killer (NK)- and T-cell markers. We have reported that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft-versus-host disease (GVHD) with retention of antitumor activity mediated by NKG2D, which is an activating receptor expressed on NK cells. The mechanism of suppression of GVHD with CIK cells is not fully understood. Host residual dendritic cells (DCs) are most important cells in initiating GVHD reaction. Therefore, we hypothesized that alloreactive NK cells, even when infused in large numbers, do not cause GVHD by killing recipient DCs with a same mechanism as CIK cells kill tumor cells. To test this, DCs generated from rodent bone marrow cells were used for 51-Cr release cytotoxicity assays as target cells. We demonstrated that though autologous CIK cells (Balb/c) had relatively strong killing activity against mature DCs (Balb/c), allogeneic CIK cells (B6) had much more killing activity even from at a 10:1 effector -target ratio as shown in Fig 1 below. In addition, killing activity against DCs did not changed with/without adding NKG2D blocking antibody, suggesting that there were possible mechanisms for cell killing other than NKG2D/NKG2D ligand system in CIK cells. To further evaluate whether allogeneic CIK cells eliminate host DCs to reduce GVHD in vivo, lethally irradiated Balb/c recipients were given BM (B6) with CIK cells or splenocytes to compare the number and activation status of residual host-typed DCs in the spleen after bone marrow transplantation. As shown in Fig 2, on day 1 after BMT absolute number of host DCs in spleen receiving splenocytes and CIK cells were almost same, however, the numbers of DCs in the mice receiving BM alone were much less. On day 3, absolute number of host DCs in all groups decreased. In particular, the number of host DCs in the mice receiving CIK cells dramatically decreased and finally reached to the same number of host DCs in the mice receiving BM alone. On the contrary, the number of host DCs in the mice receiving splenocytes didn't decrease so much and was significantly greater than number of host DCs in the mice receiving BM alone. Considering the above, when allo-CIK cells first met to DCs, both DCs and CIK cells were expanded on day 1 after BMT, and then allo-CIK cells turned to kill host DCs. In conclusion, allogeneic CIK cells caused less GVHD due in part to elimination of host DCs. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Bone Marrow Cavity Is a Feasible Site for Islet Transplantation in Nonhuman Primates

Introduction: The liver, which is currently the only approved site for clinical islet transplantation, presents numerous immunologic and non-immunologic barriers to islet engraftment and survival. The bone marrow cavity represents an attractive alternative site: It avoids liver-specific first-pass metabolism, is easily accessed, and intraosseous delivery has successfully been used as an alternative to intravenous delivery for human bone marrow transplantation. In light of the successful transplantation of islets into the bone marrow of rodents, we hypothesized that the bone marrow could be a feasible site for islet transplantation in nonhuman primates. Methods: Five rhesus macaques were rendered diabetic with streptozotocin. A mean of 13,991 (±3,114) MHC-mismatched islet equivalents per kilogram were transplanted into the bone marrow cavity at five separate sites, including bilateral humeri, bilateral femurs and the iliac crest. Recipients were treated with an immunosuppression regimen that we have previously shown significantly prolongs islet allograft survival: induction therapy with TS1/22, a monoclonal antibody to LFA-1, and belatacept maintenance therapy. Rejection was defined as fasting blood glucose greater than 130mg/dL on two consecutive days. Results: Four of five recipients achieved immediate insulin-independent normoglycemia. The fifth recipient required insulin therapy to maintain normoglycemia; this rate of engraftment with insulin independence is similar to that seen with intraportal islet transplantation. Rejection-free survival for recipients with functional grafts is currently 80, >79, >51 and >29 days. Conclusion: The bone marrow cavity may be a feasible alternative to intraportal islet transplantation and provide an attractive option for clinical islet transplantation.

Mesenchymal stromal cell cryopreservation.

The advent of stem cells and stem cell-based therapies for specific diseases requires particular knowledge of laboratory procedures, which not only guarantee the continuous production of cells, but also provide them an identity and integrity as close as possible to their origin. Their cryopreservation at temperatures below -80°C and typically below -140°C is of paramount importance. This target can be achieved by incorporating high molar concentrations of cryoprotectant mixtures that preserve cells from deleterious ice crystal formation. Usually, dimethyl sulfoxide (DMSO) and animal proteins are used as protectant reagents, but unexpected changes in stem cell fate and downstream toxicity effects have been reported, limiting their wide use in clinical settings. In scientific reviews, there are not much data regarding viability of mesenchymal stromal cells (MSCs) after the freezing/thawing process. During our routine analysis, a poor resistance to cryopreservation of these cells was observed, as well as their weak ability to replicate. This is an important point in the study of MSCs; moreover, it represents a limit for preservation and long-term storage. For this reason, MSCs isolated from equine, ovine, and rodent bone marrow and equine adipose tissue were compared using different cryopreservation solutions for this study of vitality. Our findings showed the best results regarding cell viability using a solution of fetal bovine serum with addition of 10% DMSO. In particular, we noted an increase in survival of equine bone marrow MSCs. This parameter has been evaluated by Trypan blue staining at fixed times (0, 24, and 48 hours post-thaw). This result highlights the fact that equine bone marrow MSCs are the frailest we analyzed. Therefore, it could be useful to delve further into this topic in order to improve the storage possibility for these cells and their potential use in cell-based therapies.

Mutagenicity and clastogenicity evaluation of tacrine by Ames and micronucleus assays

Tacrine was evaluated for its mutagenic and clastogenic activities using the Ames bacterial reverse-mutation assay and the rodent bone marrow micronucleus assay. Tacrine was tested for mutagenic potential at six different concentrations, with 1,250 µg/plate as the highest concentration, followed by five lower concentrations with 2-fold spacing. In clastogenic evaluation, tacrine was administered orally to Wistar rats for 2 days at 5, 10, and 20 mg/kg body weights to assess micronucleus induction in bone marrow erythrocytes. In the Ames assay, tacrine showed nonmutagenicity in four tester strains of Salmonella typhimurium viz. TA98, TA100, TA102, and TA1535, but it was found to be mutagenic in the TA1537 tester strain, both in the presence and absence of a metabolic activation system. Tacrine was found to be nonclastogenic on bone marrow cells of rats at all doses tested and was found to be mutagenic in only the TA1537 strain of Salmonella.