Cell and Tissue Banking

Effect of antibiotic impregnation time on the release of gentamicin from cryopreserved allograft bone chips: an in vitro study Abstract Freezing is the most common method for storing bones until use in skeletal reconstruction. However, the effect of freezing at different temperatures on antibiotic delivery from antibiotic-coated bone chips has not been evaluated. In this study, we compared antibiotic delivery in vitro from gentamicin-coated human bone stored at different temperatures impregnated for different time periods. Bone chips obtained from human femur heads were chemically cleaned and mixed with gentamicin sulfate solution for 1 h and 10 h respectively. Samples of both groups were cryopreserved for 4 months at − 20 °C, 4 months at − 80 °C, or evaluated immediately without freezing. Antibiotic release from the bone chips was measured using Bacillus subtilis as an indicator strain. Zones of inhibition and rates of gentamicin were higher for the samples impregnated for 10 h as compared to 1 h. There was no significant difference between non-cryopreservation, cryopreservation at different temperatures of − 20 and − 80 °C on the release of gentamicin from bone chips even after storage for 4 months.

Correction to: Defined serum- and xeno-free cryopreservation of mesenchymal stem cells In the original article, Fig. 1A was by mistakenly duplicated. The corrected image is provided in this correction article.

Comparative evaluation of bioburden and sterility of indigenously prepared bone allograft with and without gentamicin Abstract During bone allograft processing, despite stringent donor screening and use of aseptic techniques, microbial invasion may occur due to the porous nature of the graft and cause potentially fatal infections. The aim of the present study was to prepare bone allograft with and without gentamicin and to compare bioburden and sterility in the obtained grafts to evaluate the role of antibiotic in enhancing graft safety. Fifty samples of demineralized freeze-dried bone allograft were prepared from suitable donors according to international standards. Randomly selected 25 samples were placed in 8 mg gentamicin/gram bone solution for 1 h. Packaging and sealing was done to ensure no microbial ingress during transportation. 40 samples were selected for bioburden testing. Remaining 10 were subjected to 25 kGy gamma radiation and tested for sterility. Microbiological evaluation revealed no evidence of colony forming units in all the samples of both the groups (Bioburden = 0). Post-radiation sterility testing also revealed no bacterial colony in the tested samples from both the groups. Favorable results validate the processing protocol while comparable results in both groups indicate no additive benefit of gentamicin addition. Nil bioburden may be used in further studies to determine a lower radiation dose to achieve adequate sterility and minimize the disadvantages of radiation like collagen cross-linking and decreased osteoinductive capacity.

Quality assessment on the long-term cryopreservation and nucleic acids extraction processes implemented in the andalusian public biobank Abstract Human samples are commonly collected and long-term stored in biobanks for current and future analyses. Even though techniques for freezing human blood are well established, the storage time can compromise the cell viability as well as the yield and quality of nucleic acids (RNA and DNA) extracted from them. In this study, a protocol to obtain peripheral blood mononuclear cells (PBMCs) from 70 subjects, which were stored at − 196 °C from EDTA tubes for a long-term, was assessed. In parallel; a protocol to obtain DNA from the same subjects, which were stored at − 80 °C from citrate tubes, was also studied. Samples stored from 2008 to 2012 were studied and the results obtained showed that there were no statistically significant differences in the RNA or DNA extracted in terms of purity, integrity and functionality The freezing protocol used by the Málaga Biobank shows that viable PBMCs and DNA could be kept for a period of, at least, 10 years, with a high quality and performance. Furthermore, RNA extracted from these PBMCs presents also a good quality and performance. Therefore, the samples frozen according to the conditions of the protocols assessed in this study could be optimal for biomedical research.

Mechanical and structural properties of human aortic and pulmonary allografts do not deteriorate in the first 10 years of cryopreservation and storage in nitrogen Abstract The aortic and pulmonary allograft heart valves (AHV) are used in the cardiac surgery for replacing the impaired semilunar valves. They are harvested from donor hearts and cryostored in tissue banks. The expiration period was set to 5 years arbitrarily. We hypothesized that their mechanical and structural properties do not deteriorate after this period. A total of 64 human AHV (31 aortic and 33 pulmonary) of different length of cryopreservation (fresh, 0–5, 5–10, over 10 years) were sampled to different tissue strips (artery, leaflet, ventriculo-arterial junction) and tested by tensile test with loading velocity 10 mm/min until tissue rupture. Neighbouring regions of tissue were processed histologically and evaluated for elastin and collagen area fraction. The results were evaluated statistically. In aortic AHV, the physical deformation response of wall samples to stress did not changed significantly neither during the process of cryopreservation nor during the first 10 years of storage. In pulmonary AHV, the ultimate strain dropped after 5 years of cryopreservation indicating that pulmonary artery was significantly less deformable at the time of rupture. On the other hand, the ultimate stress was equal during the first 10 years of cryostorage. The changes in collagen and elastin amount in the tissue samples were not associated with mechanical impairment. Neither elasticity, stiffness and solidity nor morphology of aortic and pulmonary AHV did not change reasonably with cryopreservation and in the first 10 years of cryostorage. This evidence suggests that the expiration period might be extended in the future.

A protocol for isolation and identification and comparative characterization of primary osteoblasts from mouse and rat calvaria Abstract Calvaria from neonatal mouse and rat is ideal resource for osteoblasts but can be easily contaminated by other cells such as fibroblasts. Here, we established a protocol for isolation and purification of primary osteoblast by enzyme sequential digestion and differential adhesion. In addition, we compared the phenotypic and functional traits of osteoblasts from mouse and rat which are commonly employed in studies. The method applied equally to rat and mouse in osteoblasts isolation and was corroborated its feasibility and validity. The results also provided us evidences for other experiments such as choosing a certain time point to give intervention and do the relevant tests.

Integration of C-type natriuretic peptide gene-modified bone marrow mesenchymal stem cells with chitosan/silk fibroin scaffolds as a promising strategy for articular cartilage regeneration Abstract The treatment of articular cartilage defects has become a major clinical concern. Currently, additional efforts are necessary to develop effective methods to cure this disease. In this work, we combined gene therapy with tissue engineering methods to test their effect on cartilage repair. In in vitro experiments, we obtained C-type natriuretic peptide (CNP) gene-modified bone marrow-derived mesenchymal stem cells (BMSCs) by transfection with recombinant adenovirus containing the CNP gene and revealed that CNP gene-modified BMSCs had good chondrogenic differentiation ability. By the freeze-drying method, we successfully synthesized a chitosan/silk fibroin (CS/SF) porous scaffold, which had a suitable aperture size for chondrogenesis. Then, we loaded CNP gene-modified BMSCs onto CS/SF scaffolds and tested their effect on repairing full-thickness cartilage defects in rat joints. The gross morphology and histology examination results showed that the composite of the CNP gene-modified BMSCs and CS/SF scaffolds had better repair effects than those of the other three groups at each time point. Additionally, compared to the group with BMSCs and scaffolds, we found that there was more cartilage matrix in the CNP gene-modified BMSCs and CS/SF scaffolds group. Data obtained in the present study suggest that the composite of CNP gene-modified BMSCs and CS/SF scaffolds represent promising strategies for repairing focal cartilage lesions.

Adipose-derived mesenchymal stem cell exosomes: a novel pathway for tissues repair Abstract The well-characterized curative effect of transplanted mesenchymal stem cells has been mainly attributed to their homing and subsequent differentiation for the repair and regeneration of damaged tissue. Adipose-derived mesenchymal stem cells (ADMSCs) are not only multipotent and plastic, but also abundant as they can be easily harvested with minimally invasive surgical techniques. This makes ADMSCs conducive for clinical applications. Recently, the secretory function of ADMSCs has been regarded as the primary mediator of MSC-based therapy. Exosomes are one kind of small cell extracellular membrane vesicles, which are primarily used to deliver cell-specific proteins, as well as nucleic acids secreted by various cell types. This review will introduce and characterize exosomes-derived ADMSCs (ADMSCs-Exo) and look at new therapies and prospective, including the limitations and outlook for therapeutic strategy. We will describe the latest research progress on myocardial repair, neuroprotection and neurotrophic effects, hepatic repair, renal repair, cutaneous repair, regeneration and other aspects using these cells.

Encapsulated explant in novel low shear perfusion bioreactor improve cell isolation, expansion and colony forming unit Abstract One of most important issue in the field of regenerative medicine is selection of appropriate cells, scaffolds and bioreactors. The present study aimed to investigate the appropriate method for the isolation of human UC-MSCs cells from explant cultured in alginate scaffold within novel perfusion bioreactor. MSCs were isolated with explant method and CD markers such CD73, CD31, CD90 and CD105 as were analyzed by flow cytometry. The culture chamber of the novel perfusion bioreactor was made from Plexiglas and housed the cell/scaffold constructs in the central part and the medium for the whole culture period. The flow behavior within the bioreactor chamber were performed for closed and open bypass systems. The shear stress profiles simulated using CFD modeling. The fluid flow distribution within the bioreactor chamber was performed in PBS solution containing a blue colorant. UC explants were resuspended in sodium alginate and were allowed to polymerize and placed in the perfusion bioreactor and cultured. MSCs were positive for mesenchymal markers such as CD73 and CD31. All 3D Perfusion bioreactor parts, except peristaltic pump was sterilizable by autoclaving. Results of CFD indicated very low wall shear stress on surface of culture chamber at flow rate 2 ml/min. The maximum wall shear stress was 1.10 × 10−3 m/s = 0.0110 dyne/cm2 (1 Pa = 10 dyne/cm2). The fluid flow distribution within the alginate gel initially exhibited oscillation. In comparison, when encapsulated explants were placed in the perfusion bioreactor, cell proliferation appeared faster (4.6 × 1011 ± 9.2 × 1011) than explants cultures in 2D conventional culture method (3.2 × 1011 ± 1 × 1011). Proliferated cell formed several colonies. Migration of chondrocytes to the periphery of the alginate bead was visible after 1 week of culture. Perfusion bioreactor with low shear stress and alginate hydrogel improve cell isolation and expansion and eliminate cell passaging and enhance colony forming unit of UC-MSCs.

Therapeutic abortion and ectopic pregnancy: alternative sources for fetal stem cell research and therapy in Iran as an Islamic country Abstract Regenerative medicine as a background of stem cell research and therapy has a long history. A wide variety of diseases including Parkinson's disease, heart diseases, multiple sclerosis, spinal cord injury, diabetes mellitus and etc. are candidate to be treated using different types of stem cells. There are several sources of stem cells such as bone marrow, umbilical cord, peripheral blood, germ cells and the embryo/fetus tissues. Fetal stem cells (FSCs) and embryonic stem cells (ESCs) have been described as the most potent stem cell source. Although their pluri- or multipotent properties leads to promising reports for their clinical applications, owning to some ethical and legal obstacles in different communities such as Muslim countries, care should be taken for therapeutic applications of FSCs and ESCs. Derivation of these cell types needs termination of pregnancy and embryo or fetus life that is prohibited according to almost all rules and teaches in Muslim communities. Abortion and termination of pregnancy under a normal condition for the procurement of stem cell materials is forbidden by nearly all the major world religions such as Islam. Legislated laws in the most of Muslim countries permit termination of pregnancy and abortion only when the life of the mother is severely threatened or when continuing pregnancy may lead to the birth of a mentally retarded, genetically or anatomically malformed child. Based on the rules and conditions in Islamic countries, finding an alternative and biologically normal source for embryonic or fetal stem cell isolation will be too difficult. On the one hand, Muslim scientists have the feasibility for finding of genetically and anatomically normal embryonic or fetal stem cell sources for research or therapy, but on the other hand they should adhere to the law and related regional and local rules in all parts of their investigation. The authors suggest that the utilization of ectopic pregnancy (EP) conceptus, extra-embryonic tissues, and therapeutic abortion materials as a valuable source of stem cells for research and medical purposes can overcome limitations associated with finding the appropriate stem cell source. Pregnancy termination because of the mentioned subjects is accepted by almost all Islamic laws because of maternal lifesaving. Also, there are no ethical or legal obstacles in the use of extra-embryonic or EP derived tissues which lead to candidate FSCs as a valuable source for stem cell researches and therapeutic applications.