Alteration of extracellular cation concentrations and ratios in culture medium does not affect first cleavage division of hamster zygotes in vitro nor overcome the 'two-cell block'

1989 ◽  
Vol 1 (3) ◽  
pp. 231 ◽  
Author(s):  
BD Bavister ◽  
M Golden
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Cell Block ◽  
Cleavage Division ◽  
Cell Stage ◽  
Wide Range ◽  
Standard Culture ◽  
Simple Medium

In vivo fertilized hamster one-cell eggs (embryos) were cultured in a simple medium that was modified to provide a wide range of concentrations and ratios of the four major cation components (sodium, potassium, calcium and magnesium) while maintaining total osmotic pressure at 290 +/- 5 mosm. Embryos were cultured in these media to find the optimum cation concentrations for supporting the first cleavage division in vitro and to determine if physiologically abnormal cation concentrations and/or ratios in standard culture media could account for the 'two-cell block' to development in vitro in this species. Despite using a broad range of ratios for sodium:potassium (from 45:1 to 5:1) and for calcium:magnesium (from 17:1 to 1:1), there were no significant differences in the proportions of fertilized eggs that underwent the first cleavage division (approx. 60-80% across all treatments), and none of the two-cell embryos underwent further cleavage during extended culture. These data demonstrate that the first cleavage division of hamster embryos in vitro is insensitive to extracellular concentrations and ratios of the major cations, and that the non-physiological concentrations and/or ratios of these cations in the culture medium are not the primary reason for the failure of hamster zygotes to develop past the two-cell stage in vitro.

L-methionine uptake, incorporation and effects on proliferative activity and protein synthesis in bovine claw tissue explants in vitro

2007 ◽  
Vol 146 (1) ◽  
pp. 103-115 ◽  
Author(s):  
N. L. HEPBURN ◽  
C. H. KNIGHT ◽  
C. J. WILDE ◽  
K. A. K. HENDRY ◽  
H. GALBRAITH
Keyword(s):  
Protein Synthesis ◽  
Culture Medium ◽  
Culture Media ◽  
Tissue Level ◽  
Normal Function ◽  
Regulation Of Proliferation ◽  
Methionine Uptake ◽  
Dna And Protein Synthesis

SUMMARYL-methionine is a sulphur-containing nutritionally essential amino acid. It has a number of important roles in epidermal and dermal tissues of the integument of animals. Failure of normal function of these tissues in the hoof (claw) is a cause of lameness in cattle. Little is known about quantitative relationships between post-absorptive concentrations of nutrients including sulphur-containing amino acids and uptake and utilization by epidermis and dermis of the bovine claw. These parameters were studied at the tissue level by use of an established in vitro claw explant system using tissue from cattle of beef or dairy origin and L-[35S]-labelled methionine as tracer. The results showed that uptake of L-methionine by freshly prepared solear explants in Dulbecco's Modified Eagle Medium/F-12 Nutrient Mix (DMEM/F12) (1:1) medium containing 1·0 mmol L-methionine/litre was concentrative after 5–8 min, essentially linear for up to 10 min and became curvilinear thereafter. Maximum uptake and steady state conditions were obtained at approximately 30 min. Further measurements were made following 21 h incubation in culture medium. Under conditions of varying concentrations of L-methionine and measurement of uptake after 30 min, the presence of a saturable curve, that obeyed Michaelis–Menten kinetics, was demonstrated. Values of 3·61 mmol/litre and 5·84 mmol/kg intracellular water/30 min were obtained for KM and Vmax, respectively. Uptake was not influenced by L-cysteine and L-cystine concentrations in the culture media.Similar culture and incubation conditions were used in subsequent studies of DNA and protein synthesis. These showed that rates of incorporation of L-methionine into protein fractions and stimulation of DNA synthesis measured by methyl-thymidine incorporation were dependent on L-methionine concentrations in the medium. Maximal rates occurred at approximately 50 μmol/litre, which is in the normal physiological range, and at 1% of maximum uptake capacity. Examination of histological sections by autoradiography showed localization of L-[35S]-labelled methionine in basal and suprabasal epidermal cells with limited retention in dermis. Measurement, by a range of histological, immunohistochemical, electrophoretic, western blotting and autoradiographic techniques, provided further evidence of L-methionine-dependent regulation of proliferation, differentiation and synthesis of proteins under physiological concentrations, by epidermal horn-forming cells.A key role for L-methionine is suggested in the production of horn in bovine claw. The extrapolation of these in vitro data provides guidance for strategies to optimize methionine supply to claw tissues in vivo. Such extrapolation suggests the appropriateness of delivery of systemic concentrations of 50 μmol L-methionine/litre to maximize proliferative and protein depositional activity in solear epidermis and dermis in vivo.

scholarly journals IN VITRO CALLUS INDUCTION AND COMPARATIVE GC-MS ANALYSIS OF METHANOLIC EXTRACTS OF CALLUS AND LEAF SAMPLES OF AMPELOCISSUS LATIFOLIA (ROXB.) PLANCH

2018 ◽  
Vol 10 (9) ◽  
pp. 68
Author(s):  
Deep Chhavi Anand ◽  
Rishikesh Meena ◽  
Vidya Patni
Keyword(s):  
Callus Induction ◽  
Culture Media ◽  
Biological Activities ◽  
Gas Chromatography Mass Spectrometry ◽  
Dodecanoic Acid ◽  
Stem Explants ◽  
Ms Analysis ◽  
Wide Range

Objective: The aim of the present study was to develop a callus induction protocol and comparative study of therapeutic phytochemicals present in in vivo leaf and in vitro callus extracts through Gas Chromatography-Mass Spectrometry analysis.Methods: Murashige and Skoog media was used as culture media for callus induction. In vitro callus induction protocol was developed by studying the effects of various plant growth regulators like auxin, 2, 4-D (2,4-dichlorophenoxyacetic acid), NAA (naphthalic acetic acid), alone and in combination with cytokinin BAP (benzyl aminopurine), on leaf and stem explants. The GC-MS analysis of Ampelocissus latifolia was carried out on Shimadzu QP-2010 plus with thermal desorption system TD 20 to study the phytochemical profile.Results: In vitro callus induction protocol was developed for the plant and callusing was done from leaf and stem explants of Ampelocissus latifolia. The best result for callus induction was obtained using leaf explant, and callus production were maximum in Murashige and Skoog medium fortified with BAP (0.5 mg/l) and NAA (1.0 mg/l). Major compounds identified in the GC-MS analysis were Campesterol, Stigmasterol, Beta-Sitosterol, Docosanol, Dodecanoic acid, etc., in in vitro extract and Beta Sitosterol, Tocopherol, Squalene, Bergamot oil, Margarinic acid, Hexadecanoic acid, etc., in in vivo extract. The different active phytochemicals identified have been found to possess a wide range of biological activities, thus this analysis forms a basis for the biological characterization and importance of the compounds identified for human benefits.Conclusion: This is the first report on callus induction in Ampelocissus latifolia. From the results obtained through the in vitro callus induction and its comparative GCMS analysis with in vivo extract, it is revealed that Ampelocissus latifolia contains various bioactive compounds that are of importance for phytopharmaceutical uses. The GCMS analysis revealed that the amount of Beta-sitosterol and 5-Hydroxymethylfurfural (HMF) was very high in in vitro extract as compared to in vivo extract.

Analysis of clonal restriction of cell mingling in Xenopus

1985 ◽  
Vol 312 (1153) ◽  
pp. 57-65 ◽  
Keyword(s):  
Horseradish Peroxidase ◽  
Cell Types ◽  
Cell Group ◽  
Cell Stage ◽  
Wide Range ◽  
Cell Groups ◽  
Cell Layers ◽  
Single Blastomeres

Cell fates were traced by injecting horseradish peroxidase into single blastomeres of Xenopus embryos at 2- to 512-cell stages. At later stages the number, types and locations of all labelled progeny were observed. Progeny of a single labelled ancestral cell divided coherently until the 12th cell generation, the onset of gastrulation, and then dispersed and mingled with unlabelled cells. Cell mingling was restricted at mediolateral and anterior—posterior boundaries. These boundaries were always respected by progeny of any blastomere labelled at the 512-cell stage but they were frequently crossed by progeny of blastomeres labelled at the 256-cell or earlier stages. The boundaries defined seven morphological compartments each populated exclusively by a group of ancestral cells at the 512-cell stage. Each blastomere that contributed progeny to the nervous system also gave rise to a wide range of cell types in all three primary germ cell layers but the clone was restricted to a single compartment. Analysis of clonal restriction of cell mingling was done in vitro . Twenty to thirty blastomeres were excised from one ancestral cell group at the 512-cell stage and combined in vitro with 20-30 blastomeres from another group. One group of blastomeres labelled with horseradish peroxidase was placed in contact with another group of unlabelled blastomeres, maintained in vitro for up to 2 days, and then processed histologically to show the distribution of labelled and unlabelled cells. Mingling was significantly greater in combinations of two of the same ancestral cell groups than in combinations of two different ancestral cell groups. A similar result was observed when a single labelled cell was combined with either the same or different ancestral cells. In all experiments the cells were significantly larger in combinations of different ancestral cell groups, indicating that they had undergone fewer divisions. These results are consistent with the hypothesis that boundaries observed in vivo are lines of clonal restriction formed by mutual inhibition of cell motility and cell division following contact between progeny of different ancestral cell groups.

scholarly journals New in vitro model to evaluate kinetics of antimycobacterial drug release from bioresorbable polymeric carriers

2020 ◽  
pp. 10-15
Author(s):  
SN Andreevskaya ◽  
TG Smirnova ◽  
EN Antonov ◽  
LN Chernousova ◽  
SE Bogorodsky ◽  
...  
Keyword(s):  
Drug Release ◽  
Culture Medium ◽  
Culture Media ◽  
Release Kinetics ◽  
Prolonged Release ◽  
Wide Range ◽  
Polymeric Carriers ◽  
The Matrix ◽  
Kinetics Of

Sustained-release drugs against tuberculosis are a promising approach to therapy since they positively affect patient compliance with long regimens, especially when it comes to the multidrug-resistant form of the disease. Conventional UV-visible spectroscopy does not work well with multicomponential culture media used for growing M. tuberculosis. The aim of this study was to develop a method for evaluating the kinetics of anti-tuberculosis drug released from bioresorbable polymeric carriers suitable for screening a wide range of encapsulated prolonged-release drugs and identifying the best performing candidate. While studying the growth dynamics of the laboratory susceptible strain M. tuberculosis H37Rv in the presence of different levofloxacin concentrations (from 0.03 to 0.4 μg/ml), we developed a model, which is essentially a set of 2 parallel experiments evaluating the kinetics of drug release into the culture medium. The results of these 2 experiments conducted on 3 encapsulated forms of levofloxacin loaded onto bioresorbable polymeric PLGA carriers (particles sized 50 μm and 100 μm and the matrix) revealed that release kinetics of the drug largely depended on the type of polymeric carrier. The best encapsulation of the antibiotic and its gradual release into the culture medium was observed for the matrix. All experiments were run in 3 replicates. The obtained data were analyzed using descriptive statistics.

191 ALLEVIATION OF THE TWO-CELL BLOCK OF ICR, ddY, AND AKR MOUSE EMBRYOS BY BOVINE SERUM ALBUMIN

2007 ◽  
Vol 19 (1) ◽  
pp. 212
Author(s):  
K. Ono ◽  
R. Ohishi ◽  
H. Imai ◽  
M. Yamada
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Culture Media ◽  
Mouse Embryos ◽  
Control Group ◽  
Cell Block ◽  
Cell Stage ◽  
Developmental Rates

Bovine serum albumin (BSA) is an embryotrophic macromolecule used in embryo culture media and improves embryo development in vitro. However, when 1-cell embryos from some strains of mouse were cultured in traditional medium, even with BSA, developmental arrest occurred at the 2-cell stage, termed '2-cell block'. The developmental block is known to be alleviated by adding EDTA to the medium for ICR and ddY strains, and deleting phosphate from the medium for the AKR strain. Recently, our preliminary experiments revealed that the 2-cell block is relieved by adding deionized BSA (d-BSA) to the medium for the ICR strain. Thus, in the present study, we investigated whether d-BSA could rescue the embryos from ICR, ddY, and AKR strains from the 2-cell block. Fertilized 1-cell embryos were collected 20 h post-hCG from superovulated ICR, ddY, and AKR females (8-week-old) that had been mated with the ICR strain of males. Stock solutions (15%) of commercially available fraction V BSA, ovalbumin (ova), and γ-globulin (γG) were deionized over a mixed-bed ion adsorption resin. Embryos were cultured in EDTA-depleted KSOM medium with or without these deionized or non-deionized proteins at 37�C under 5% CO2 in air for 4 days. Experiments were done in at least 3 replicates, and the statistical analyses of the data were done by ANOVA and Fisher's PLDS test. To observe the distribution of BSA in the embryos from the 1-cell to the blastocyst stages, immunofluorescence study was performed using anti-BSA antibody with a laser confocal microscope. The developmental rates to the 4-cell stage of 1-cell embryos cultured in medium without (control group) or with BSA at 0.3% in ICR and 0.6% in ddY and AKR (BSA group) were very low (ICR: 10% (4/38) and 37% (17/47); ddY: 9% (7/73) and 23% (9/37); AKR: 0% (0/60) and 0% (18/30), respectively). However, when embryos were cultured with d-BSA at 0.3% in ICR and 0.6% in ddY and AKR, the rates to the 4-cell stage significantly increased (ICR: 91% (51/56), ddY: 82% (61/76), AKR: 82% (50/60) vs. control group or BSA group: P < 0.05), and development to the blastocyst stage was observed (ICR: 79% (44/56), ddY: 65% (47/76), AKR: 63% (38/60)). When ICR embryos were cultured with 0.3% deionized-ova or deionized-�G, no significant increase was observed in developmental rates to the 4-cell stage (25% (10/40) and 24% (10/42), respectively). We next examined the critical culture period for the beneficial effects of d-BSA and intracellular distribution of BSA using ddY mouse embryos. It was found that exposure to d-BSA during the late 1-cell (24 h post-hCG) and early 2-cell stages (42 h post-hCG) promoted the development beyond the 2-cell stage. The distribution of BSA in the cytoplasm of embryos at any stage was observed. Interestingly, BSA localized in the nuclei of embryos during the late 1-cell and early 2-cell stages. In conclusion, our results suggest that BSA itself has a potential to remove the 2-cell block in ICR, ddY, and AKR strains. In addition, nuclear localization of BSA may play a key role in regulating the development beyond the 2-cell stage in the mouse embryos.

scholarly journals In vitro culture of Neoechinorhynchus buttnerae (Acanthocephala: Neoechinorhynchidae): Influence of temperature and culture media

2018 ◽  
Vol 27 (4) ◽  
pp. 562-569 ◽  
Author(s):  
Carinne Moreira de Souza Costa ◽  
Talissa Beatriz Costa Lima ◽  
Matheus Gomes da Cruz ◽  
Daniela Volcan Almeida ◽  
Maurício Laterça Martins ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Treatment Strategies ◽  
Infected Fish ◽  
In Vivo Studies ◽  
Colossoma Macropomum ◽  
Tank Water

Abstract Infection by the acantocephalan Neoechinorhynchus buttnerae is considered one of most important concerns for tambaqui fish (Colossoma macropomum ) production. Treatment strategies have been the focus of several in vivo studies; however, few studies have been undertaken on in vitro protocols for parasite maintenance. The aim of the present study was to develop the best in vitro culture condition for N. buttnerae to ensure its survival and adaptation out of the host to allow for the testing of substances to be used to control the parasite. To achieve this, parasites were collected from naturally infected fish and distributed in 6-well culture plates under the following treatments in triplicate: 0.9% NaCl, sterile tank water, L-15 Leibovitz culture medium, L-15 Leibovitz + agar 2% culture medium, RPMI 1640 culture medium, and RPMI 1640 + agar 2% culture medium. The plates containing the parasites were maintained at 24 °C, 28 °C, and 32 °C. The RPMI 1640 + agar 2% culture medium showed the best survival of 24 days at 24 °C. No body alterations such as swollen parasites, body deformation, dehydration and hardening were observed in the RPMI 1640 + 2% culture medium.

Analysis of specific factors generating 2-cell block in AKR mouse embryos

Zygote ◽  
2006 ◽  
Vol 14 (2) ◽  
pp. 169-179 ◽  
Author(s):  
Akihiro Yoneda ◽  
Aki Okada ◽  
Teruhiko Wakayama ◽  
Junji Ueda ◽  
Tomomasa Watanabe
Keyword(s):  
Mouse Strain ◽  
Culture Medium ◽  
Mouse Strains ◽  
C57bl Mouse ◽  
Cell Block ◽  
Cell Stage ◽  
Female Progeny ◽  
Reconstructed Embryos ◽  
Specific Factors

SummaryThe phenomenon of the developmental arrest at the 2-cell stage of 1-cell embryos from some mouse strains during in vitro culture is known as the 2-cell block. We investigated the specific factors involved in the 2-cell block of AKR embryos by means of a modified culture system, the production of reconstructed embryos by pronuclear exchange and a cross experiment. In a culture medium with phosphate, 94.6% of 1-cell embryos from the C57BL mouse strain developed to the blastocyst stage, but 95.7% of embryos from the AKR mouse strain showed 2-cell block. Phosphate-free culture medium rescued the 2-cell block of AKR embryos and accelerated the first cell cycle of the embryos. Co-culture with BRL cells and a BRL-conditioned medium fractionated below 30 kDa also rescued the 2-cell block of AKR embryos. Examinations of in vitro development of reconstructed embryos and of embryos from F1 females between AKR and C57BL strains clearly demonstrated that the AKR cytoplast caused the 2-cell block. In the backcrossed female progeny between (AKR × C57BL) F1 males and AKR females, about three-quarters of the embryos were of the 2-cell blocking phenotype and about one-quarter were of the non-blocking phenotype. These results suggest that two genes are responsible for the 2-cell block of AKR embryos.

scholarly journals 248 EMBRYO DEVELOPMENT IN MICRODROPS AND MICROCHANNELS: COMPARISON OF NCSU23 SEQUENTIAL AND NON-SEQUENTIAL CULTURE MEDIUM

2005 ◽  
Vol 17 (2) ◽  
pp. 274
Author(s):  
A.S. Lima ◽  
C.E. Ferguson ◽  
M.B. Wheeler
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Culture Media ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
The Other ◽  
Cell Stage ◽  
Development Rates ◽  
Hatching Rates

The in vitro culture systems used to produce pig embryos generally result in few embryos developing to the blastocyst stage. The use of pyruvate (pyr) and lactate (lac) during the culture of zygotes to the 8-cell stage followed by glucose (glu) supplementation replacing pyr and lac appears to be beneficial for embryo development in the pig. The aim of this study was to compare the embryo development rates from pig oocytes fertilized with and without cumulus cells in 100-μL microdrops (MD) and cultured in 100-μL MD or microchannels (MC), using NCSU23 containing 8 mg/mL of BSA and supplemented with (1) glu or (2) pyr/lac or (3) pyr/lac for the first three days and then with just glu for the remainder of culture period (pyr/lac-glu). Sow oocytes were matured in TCM199 supplemented with gonadotropins for the first 22 h, and for an additional 22 h without hormones. After 44 h of maturation, oocytes were placed in MD of modified tris-buffered medium to be fertilized using 3 × 105 sperm/mL. Oocytes were divided into two groups for fertilization: with and without cumulus cells. Following 6 h of fertilization, all inseminated oocytes were washed, divided into groups of 15, allotted to the three culture media treatment groups as described above, and incubated in either MD or MC. With the exception of one treatment there were no significant differences in development rates among embryos cultured in MD or MC, hence data were pooled from these two culture devices. Only oocytes fertilized without cumulus cells and cultured in pyr/lac in MC appeared to have lower rates of blastocyst formation (11.67%) than those cultured in MD (26.67%) in the same culture medium. When the six treatments were compared, oocytes fertilized with cumulus cells and cultured in glu had significantly higher (P < 0.05) blastocyst rates and hatching rates compared with the other treatments, with the exception of those fertilized without cumulus cells and cultured in pyr/lac-glu. There were no significant differences among other treatments in Day 7 blastocyst or in Day 9 hatching rates. In conclusion, both culture devices can be used to reach similar blastocyst rates with different treatments. In this experiment, the removal of cumulus cells before fertilization appeared to enhance embryo development in vitro when sequential media are used. On the other hand, the presence of cumulus cells before fertilization seems to enhance embryo development when non-sequential glu medium is used. Table 1. Embryo development rates on Day 9 for three different culture treatments

140 INTERFERON-τ SECRETION BY IN VIVO DERIVED BOVINE TROPHOBLASTIC VESICLES

2010 ◽  
Vol 22 (1) ◽  
pp. 229
Author(s):  
Y. Hashiyada ◽  
H. Takahashi ◽  
M. Geshi
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Spherical Shape ◽  
Morphological Observation ◽  
Measure Concentration ◽  
Fetal Bovine ◽  
Implantation Period

In ruminants, interferon-τ (IFN-τ) is a major pregnancy factor, secreted by the embryonic trophoblast cells during the pre-implantation period, being important for the maternal-fetal recognition. The co-transfer of bovine trophoblastic vesicles (bTVs) derived from in vivo recovered conceptuses is known to promote the successful implantation of embryos with expected lower viability, such as in vitro handled embryos, through the effects of IFN-τ secreted by bTVs. We have also reported that the pregnancy rate was improved using this technique in early pregnancy phase (Hashiyada et al. 2005 J. Reprod. Dev. 51, 749-756). However, the IFN-τ secretion level from bTVs has not been well known. Therefore, the objective of the present study was to measure concentration of IFN-τ released from individually cultured bTVs in vitro. Furthermore, we also investigated the transition of IFN-τ level in continuous culture of bTVs. Blastocysts were produced by artificial insemination of Japanese black cows following superstimulatory treatment and were recovered on 16 or 18 days post-estrus. Sixty-eight bTVs were prepared from 23 elongating blastocysts, 3 to 20 mm in length, by dissection using a surgical blade. Each trophoblastic fragment, 1 to 1.5 mm in width, was cultured in a well of 96-well plates using TCM-199 supplemented with 20% (v/v) fetal bovine serum and 0.1 mM β-mercaptoethanol at 38.5°C in a humidified atmosphere of 5% CO2 in air. After 24 h of culture, fragments of unformed vesicles were re-cultured for an additional 24 h; 10 bTVs from this group were continuously cultured until Day 24 (the day of insemination was defined as Day 0). The volume of culture medium was 100 μL/well/day until Day 2 and thereafter changed to 200 μL/well/2 days to terminate. The viability of bTVs was assessed based on maintained spherical shape of vesicle, morphologically. Exchange and collection of culture media, morphological observation of bTVs were performed on Days 1, 2, 4, 8, 10, 12, 14, 16, 18, 20, 22, and 24. Culture fluids were stored at -30°C. IFN-τ was measured by RIA (Takahashi H et al. 2005 Theriogenology 63, 1050-1060). Data were analyzed by Student’s τ-test Initial IFN-τ secretion did not differ between groups that had formed and unformed vesicles on Day 1, 89.8 ± 7.1 (mean ± SEM, n = 41) and 76.6 ± 7.2 ng mL-1 (n = 27), respectively. On Day 2, in the unformed group, all of the fragments had made vesicles and the IFN-τ increased to 99.4 ± 11.8 ng mL-1. In the extended culture group (n = 10), IFN-τ secretion tended to increase from Day 2 (66.9 ± 14.2 ng mL-1) to Day 8 (166.0 ± 46.7 ng mL-1) (P = 0.06). However, this large amount of IFN-τ on Day 8 significantly decreased from Day 10 (32 ± 4.9 ng mL-1, P < 0.05) to Day 24 (9.2 ± 1.0 ng mL-1, P < 0.05) gradually. The survival rate of these bTVs decreased to 90% (9/10) on Day 10 and then to 60% (6/10) during Days 18 to 22. These results indicate that bTVs cultured for a long term in vitro might decrease IFN-τ secretion.

scholarly journals The superiority of conditioned medium derived from rapidly expanded mesenchymal stem cells for neural repair

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ya-Tzu Chen ◽  
May-Jywan Tsai ◽  
Nini Hsieh ◽  
Ming-Jei Lo ◽  
Meng-Jen Lee ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Conditioned Medium ◽  
Culture Medium ◽  
Standard Medium ◽  
Neural Repair ◽  
Cord Injury ◽  
Standard Culture ◽  
Standard Culture Medium

Abstracts Background Spinal cord injury (SCI) is a complex and severe neurological condition. Mesenchymal stem cells (MSCs) and their secreted factors show promising potential for regenerative medicine. Many studies have investigated MSC expansion efficacy of all kinds of culture medium formulations, such as growth factor-supplemented or xeno-free medium. However, very few studies have focused on the potential of human MSC (hMSC) culture medium formulations for injured spinal cord repair. In this study, we investigated the effect of hMSC-conditioned medium supplemented with bFGF, EGF, and patient plasma, namely, neural regeneration laboratory medium (NRLM), on SCI in vitro and in vivo. Methods Commercial and patient bone marrow hMSCs were obtained for cultivation in standard medium and NRLM separately. Several characteristics, including CD marker expression, differentiation, and growth curves, were compared between MSCs cultured in standard medium and NRLM. Additionally, we investigated the effect of the conditioned medium (referred to as NRLM-CM) on neural repair, including inflammation inhibition, neurite regeneration, and spinal cord injury (SCI), and used a coculture system to detect the neural repair function of NRLM-MSCs. Results Compared to standard culture medium, NRLM-CM had superior in inflammation reduction and neurite regeneration effects in vitro and improved functional restoration in SCI rats in vivo. In comparison with standard culture medium MSCs, NRLM-MSCs proliferated faster regardless of the age of the donor. NRLM-MSCs also showed increased adipose differentiative potential and reduced CD90 expression. Both types of hMSC CM effectively enhanced injured neurite outgrowth and protected against H2O2 toxicity in spinal cord neuron cultures. Cytokine arrays performed in hMSC-CM further revealed the presence of at least 120 proteins. Among these proteins, 6 demonstrated significantly increased expression in NRLM-CM: adiponectin (Acrp30), angiogenin (ANG), HGF, NAP-2, uPAR, and IGFBP2. Conclusions The NRLM culture system provides rapid expansion effects and functional hMSCs. The superiority of the derived conditioned medium on neural repair shows potential for future clinical applications.

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