Showing 9 results for Conditioned Medium
Somayyeh Sadat Tahajjodi, Ehsan Farashahi Yazd, Azam Agha-Rahimi, Reza Aflatoonian, Mohammad Ali Khalili, Mahnaz Mohammadi, Behrouz Aflatoonian,
Volume 18, Issue 1 (1-2020)
Abstract
Background: Cumulus cells, as oocyte nurse cells, provide a suitable microenvironment with growth factors and cellular interactions required for oocyte maturation. Thus, these cells may serve as a natural niche for in vitro studies of female germ cell development. Cumulus cells may help attain a better understanding of the causes of infertility in women and eventually improve the outcomes of cases that respond poorly to standard infertility treatment.
Objective: The aim of this study was to isolate, culture, and investigate the biological characteristics of human cumulus cells.
Materials and Methods: In this experimental study, cumulus cells were isolated, cultured, and characterized using reverse transcription-polymerase chain reaction analyses of specific genes including FOXL2, CYP19A1, FSHR, AMHR, and LHR. The presence of vimentin, a structural protein, was examined via immunofluorescent staining. Moreover, levels of anti-mullerian hormone (AMH) and progesterone secretion by cumulus cells were measured with ELISA after 2, 4, 12, 24, and 48 hr of culture.
Results: In adherent culture, human cumulus cells expressed specific genes and markers as well as secreted AMH and progesterone into the medium.
Conclusion: Cumulus cells secrete AMH and progesterone in an adherent culture and might be applicable for in vitro maturation (IVM) and in vitro gametogenesis (IVG) studies.
Maryam Adib, Seyed Morteza Seifati, Mahmood Dehghani Ashkezari, Arezoo Khoradmehr, Roshan Rezaee-Ranjbar-Sardari, Somayyeh Sadat Tahajjodi, Behrouz Aflatoonian,
Volume 18, Issue 12 (12-2020)
Abstract
Background: To increase the results of infertility treatment, many efforts have been made to improve the treatment methods. As assisted reproductive technology is mainly using cell culture methods, one of the approaches to improve this technology is conditioned medium from different sources. It is desirable to apply in vitro maturation (IVM) and use oocytes from normal cycles instead of stimulating ovulation.
Objective: To investigate the effect of human cumulus cell condition medium (hCCCM) on the IVM of immature mouse oocytes and morphology.
Materials and Methods: In this experimental study, 240 germinal vesile oocytes were collected from four-six wk-old mice after 48 hr of 5IU pregnant mare serum gonadotropin (PMSG) injection and cultured in hCCCM (test group, n = 120) and DMEM + 20% FBS (control group, n = 120). The IVM rates and changes in perivitelline space (PVS) and shape were investigated at 8, 16, and 24 hr following the culture. The mature (MII) oocytes were subjected to in vitro fertilization (IVF) and the fertilization rate was assessed in three days.
Results: A significant difference was observed between the maturation rates in the hCCCM and control groups (24.16% vs 0%; p = 0.001), as well as morphologic changes between the two groups (p = 0.04, p = 0.05). The development rate for MII oocytes attained from IVM in the hCCCM group was 27.58% (2-cell) and 6.89% (4-cell). Data displayed that hCCCM is an effective medium for oocytes maturation compared to the control medium.
Conclusion: hCCCM supports oocyte in vitro growth and maturation. Moreover, hCCCM changes the oocyte shape and size of perivitelline space.
Tahajjodi S, Farashahi-Yazd E, Agha-Rahimi A, Akyash F, Hajizadeh Tafti F, Aflatoonian R, Aflatoonian B,
Volume 19, Issue 5 (5-2021)
Abstract
Background: Human embryonic stem cells (hESCs) can differentiate to germ cells as confirmed using gene expression assessments. Cumulus cells are physically closest cells to the developing oocyte and they have positive effects on oocytes maturation by secreting some factors that have a crucial role in the process of oogenesis. Thus, in vitro differentiation of embryonic stem cells may also be affected of cumulus-secreted factors.
Objective: The aim of this study is assessment the effect of the cumulus cells conditioned medium (CCCM) on differentiation of hESCs to female germ cells.
Materials and Methods: Embryoid bodies (EBs) from Yazd4-hESCs were formed and cultured for 14 days into 4 different conditions: 1) spontaneously differentiation in EB medium (SD-EB), 2) treated with 40% CCCM (CCCM-EB), 3) spontaneously differentiation in 40% DMEM+20% FBS (SD-DM), and 4) treated with 40% CCCM in DMEM+20% FBS (CCCM-DM). Expression of pluripotency and germ cells genes were examined in EBs from each group by RT-qPCR at the time of days 0, 4, 7 and 14. In addition, immunofluorescent (IF) staining was done for pluripotency and germ cells markers.
Results: RT-qPCR data revealed that CCCM-EB and CCCM-DM had a significant increase in differentiation of female germ cell from hESCs than SD-EB and SD-DM. On the other hand, comparison between basal media revealed that EB medium is a better medium than DMEM+20% FBS for female germ cell development from hESCs. Localization of the germ cells within the cultures was detected using IF for TRA-2-49, SSEA1 and VASA antibodies in all groups.
Conclusion: Cumulus cells conditioned medium supports female germ cell development from hESCs assessed by gene expression profile. Also, EB medium as basal medium has better impact on differentiation induction.
Tork S, Molaeeghaleh N, Abdi S, Movassaghi S, Sharifi Z,
Volume 19, Issue 5 (5-2021)
Abstract
Background: In vitro culture of isolated follicles of cryopreserved ovaries can be proposed as a fertility preservation method. MSCs secreting various levels of growth factors and are an appropriate option for enriching the follicle culture media.
Objective: The purpose of this study was to evaluate the effect of human bone marrow mesenchymal stem cells derived conditioned medium (hBMSCs-CM) on growth and maturation of mouse ovarian follicle, and embryonic development after vitrification.
Materials and Methods: hBMSCs were cultured and the collected conditioned medium was concentrated and stored. The collected ovaries from Two-wk-old mice were divided randomly into vitrified and non-vitrified groups. Preantral follicles of both groups were isolated and cultured in α-MEM enriched with ITS and FBS supplemented with different concentrations of CM (2.5, 5, and 7.5%) for 12 days. During the culture period, survival rate, follicular maturation, follicular diameter, and levels of 17 βestradiol secretion were evaluated. In vitro fertilization and embryonic development were observed after culture.
Results: The survival rate, antrum formation, and oocyte maturation were higher in 7.5% CM subgroups than 2.5 and 5% CM in both vitrified and non-vitrified groups. Also, the follicle diameter in 7.5% CM was higher than other subgroups of both groups on day 4. Higher percentages of fertilization and embryo development were seen in 7.5% CM subgroups of the non-vitrified and vitrified group. Also, higher hormone secretion was observed in 7.5% CM subgroup in both vitrified and non-vitrified groups.
Conclusion: The present study suggests that the addition of 7.5% CM to mice ovarian preantral follicle culture media enhances follicle growth and oocyte maturation.
Moradi M, Kalehoei E, Azadbakht M, Zhaleh H, Saghari S, Parvini M,
Volume 19, Issue 5 (5-2021)
Abstract
Background: Endometriosis is an estrogen-dependent chronic inflammatory disorder that adversely affects women in their reproductive age, inducing infertility and pelvic pain. Indeed, oocytes retrieved from endometriosis women are more likely to fail in vitro maturation (IVM), exhibit altered morphology, and lower cytoplasmic mitochondrial content. More importantly, this condition is responsible for 30% of female infertility, and between 30 and 50% of women with endometriosis experience difficulties in becoming pregnant. L-carnitine (LC) is a lysine derivative with anti-oxidative properties that clears hydrogen peroxide and products of lipid peroxidation. Repaglinide (RG) is an anti-diabetic drug that increases intracellular Ca2+concentration through opening the cells' calcium channels, resulting in insulin release from pancreatic β cells. Mesenchymal stem cell-conditioned media (MSC-CM) contain various growth factors, cytokines, bioactive factors, and tissue regenerative elements generated by mesenchymal stem cells, which can enhance IVM and subsequent embryonic development.
Objective: The current study was aimed to explore the comparative effects of mesenchymal stem MSC-CM, RG, and LC on IVM, in vitro fertilization (IVF), embryo development and formation, as well as on total blastocyst cell numbers.
Materials and Methods: Immature oocytes were collected from two groups of normal and endometriosis induced female NMRI mice ovaries (6-8 weeks old). Oocytes cultured in IVM medium supplemented with 0.0 (control group), 1μM RG, 0.3 and 0.6 mg/ml LC, and 25% and 50% MSC-CM. After 24h of oocyte incubation, the IVM rate was evaluated. Subsequently, MII oocytes were put in the IVF and culture medium then early embryo cleavage was evaluated for 1 to 5 days.
Results: Endometriosis caused a devastating impact upon ovarian histopathology, oocyte maturation, fertilization, early embryo development, and oxidant/antioxidant status (p < 0.05). Conversely, in both normal and endometriosis induced mice, different concentrations of RG, LC, and MSC-CM, especially 50% MSC-CM, significantly improved IVM, IVF, and embryo formation rates compared to control groups (p < 0.05). Strikingly, better improvement in alleviating EMS-induced injuries was seen in the MSC-CM groups in comparison with other groups.
Conclusion: By way of conclusion, supplementation of IVM medium with RG, LC, and MSC-CM improved oocyte developmental parameters such as maturation, fertilization, and embryo cleavage rates. Our findings indicated that 50% of MSC-CM was the most efficient supplement to reverse endometriosis-evoked deleterious impacts. Indeed, MSC-CM not only does it possesses anti-oxidative properties, but also it contains growth factors, bioactive factors, cytokines and performs anti-inflammatory actions. Present findings have potential applications for improving the clinical trials of humans suffering from endometriosis-related sub/infertility.
Moshrefi M, Ghasemi-Esmailabad S, Shahmohammadi S, Javaheri A, Khajehmohammadi M, Nikukar H,
Volume 19, Issue 5 (5-2021)
Abstract
Background: Todays, the new infertility treatment methods try to mimic the ovarian natural micro-environment by tissue engineering. An artificial ovary tries to preserve fertilization, even with producing oocytes or with releasing steroid hormones. Producing oocytes can be obtained even by follicle culture or by ovarian tissue culture. Many physical and chemical factors are involved in ovarian cells and follicular culture. Based on the current studies, no study has reported the best mediums for ovarian cells, follicles and tissue culture.
Objective: This study aimed to uncover the appropriate media and supplements for in vitro culture of ovarian and cumulus cells (CCs).
Materials and Methods: Cortical, medullar, and hilar cells of human ovary were cultured and their conditioned medium (CMs) were collected. The expression of GDF9 was detected in all the cells. Also, CCs were collected from healthy women, who referred due to male factor infertility. To choose the optimum basal medium, a mixture of ovarian cells was cultured with basal mediums, supplemented with various concentrations of fetal bovine serum (FBS) and human serum albumin (HSA). The cocktails were as follows: [Serum free mediums], [mediums + 10% FBS], [mediums + 20% FBS], [mediums + 1% Alb], [mediums + 2% Alb], [mediums + 10% FBS + 1% Alb], [mediums + 10% FBS + 2% Alb], [mediums + 20% FBS + 1% Alb] and [mediums + 20% FBS + 2% Alb]. The same process was repeated for CCs. Because the CCs need some supplementation, we cultured them with various concentrations of some supplements to choose the best concentration. So, CCs were cultured with various concentrations of L-Glutamine, bovine serum albumin (BSA), HSA, insulin transferrin selenium (ITS), Follitropin alfa® and Pregnyl®. Also, CCs were treated with various concentrations of follicular fluids (FFs) and CMs, too. CMs were collected from ovarian, testicular, adipose and amniotic derived and ovarian carcinoma cells. Then, CCs morphology and proliferation were evaluated.
Results: All the ovarian cells expressed GDF9, as a key factor for ovarian follicular growth. Alfa MEM + 20% FBS and DMEM F12 + 20% FBS were the most suitable cocktails for ovarian and CCs culture, respectively. 20% FBS was superior to 10% for both ovarian and CCs. Also, HSA could not support the growth of ovarian and CCs, alone. The cocktails of mediums with 20% FBS and (mediums+FBS+HSA) were superior to the others. The CMs of ovarian cortical and hilar+medullar cells could lead to higher CCs growth. 17 mM/l L-Glutamine, 24 mg/ml BSA, 20 mg/ml HSA, 10 ng/ml ITS, 300 mIU/ml Follitropin alfa and 3.5 IU/ml Pregnyl led to higher proliferation of CCs.
Conclusion: Ovarian chemical micro-environment is very complex and ovarian follicle growth needs many known and unknown elements like growth factors, which are expensive. We concluded that CMs and serums can support the follicular growth alongside with basal mediums, supplemented with hormones, ITS and L-Glutamine, which are cheaper and more accessible.
Kanadi Sumapraja, Andon Hestiantoro, Isabella Kurnia Liem, Arief Boediono, Teuku Z Jacoeb,
Volume 19, Issue 12 (12-2021)
Abstract
Background: The umbilical cord-derived mesenchymal stem cells conditioned medium (UC-MSCs-CM) produces secretomes with anti-apoptotic properties, and has the potential to prevent apoptosis of granulosa cells (GC) during controlled ovarian hyperstimulation.
Objective: To observe the effect of UC-MSCs-CM on the interaction between pro- and anti-apoptotic proteins and the influence of growth differentiation factor 9 (GDF9) production in GC.
Materials and Methods: UC-MSCs-CM was collected from umbilical cord stem cell culture on passage 4. GCs from 23 women who underwent in vitro fertilization were cultured and exposed to UC-MSCs-CM for 24 hr. Then RNA of the GC was extracted and the mRNA expression of BCL-2 associated X (BAX), survivin and GDF9 were analysed using quantitative real-time PCR. The spent culture media of the GC were collected for measurement of insulin growth factor 1 using ELISA.
Results: The expression of BAX was significantly different after UC-MSCs-CM exposure (4.09E-7 vs. 3.74E-7, p = 0.02). No significant changes occurred in survivin, BAX/survivin ratio, and GDF9 expression after UC-MSCs-CM exposure (p > 0.05). The IGF-1 level of the CM was significantly higher after the CM was used as a culture medium for GC (2.28 vs. 3.07 ± 1.72, p ≤ 0.001). A significant positive correlation was found between survivin and GDF9 (r = 0.966, p ≤ 0.001).
Conclusion: IGF-1 produced by UC-MSCs-CM can work in paracrine fashion through the IGF receptor, which can inhibit BAX and maintain GDF9 production. Moreover, under the influence of UC-MSCs-CM, GC are also capable of producing IGF-1, which can impact GC through autocrine processes.
Zeynab Esmailpour, Soheila Madadi, Maryam Baazm,
Volume 22, Issue 2 (2-2024)
Abstract
Abstract
Background: Cyclophosphamide (CP) has some negative effects on the reproductive system. Stem cells and their metabolites are being utilized to enhance fertility after chemotherapy.
Objective: This study aimed to investigate the impact of conditioned medium (CM) derived from bone marrow mesenchymal stromal stem cells (BM-MSCs) on the toxic effects of CP on testicles.
Materials and Methods: BM-MSCs were isolated, a CM was collected and 25-fold concentrated. 24 male Wistar rats (8 wk, 200-250 gr) were randomly divided into following groups: control, CP, CP+Dulbecco’s Modified Eagle Medium (DMEM), CP+CM. CP was given at a single dose of 100 mg/kg. 2 wk after the CP administration, CM was injected into the testicular efferent duct. Sperm parameters, testicular histopathology, and the level of testosterone were analyzed 2 months after treatment. The expression of B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (Bax) genes were evaluated by real-time polymerase chain reaction.
Results: CP had a negative effect on testis histology (p < 0.001) and sperm quality (p < 0.001). It changed the expression of genes associated with apoptosis (p < 0.001). Treatment with CM reduced the expression of Bax (p < 0.001), while significantly increasing the expression of Bcl2 (p = 0.01). It improved sperm count (p = 0.03), viability (p < 0.001), motility (p < 0.001), spermatogonial count (p < 0.001), and epithelial thickness of testicular tubules (p = 0.02).
Conclusion: These findings suggest that CM produced from BM-MSCs may be valuable for therapeutic approaches in reproductive medicine and may lessen the side effects of CP.
Somayyeh Sadat Tahajjodi, Ehsan Farashahi Yazd, Azam Agharahimi, Fatemeh Akyash, Fatemeh Hajizadeh-Tafti, Jalal Golzadeh, Reza Aflatoonian, Behrouz Aflatoonian,
Volume 23, Issue 1 (1-2025)
Abstract
Background: Studies have shown that embryonic stem cells can differentiate into germ cells either spontaneously or directed by the defined factors or by applying a co-culture method or conditioned medium of stromal cells.
Objective: Here, Yazd4 (human embryonic stem cells [hESC] line; 46, XX) was used to evaluate the effect of human cumulus cells conditioned medium (CCCM) on in vitro germ cell production and development.
Materials and Methods: In this experimental study 2 different complete media were applied, Dulbecco’s modified eagle’s medium + 20% fetal bovin serum and embryoid body (EB) medium (KnockOut serum replacement- hESC without basal fibroblast growth factor). Thus, there are 2 control groups for spontaneous differentiation (SD) (SD-EB and SD-DM) and 2 types of CCCM (CCCM-DM and CCCM-EB) as test groups. 40% dilution of the conditioned medium was applied according to previous reports.
Results: Reverse transcription-quantitative polymerase chain reaction and immunofluorescent staining assays revealed that between groups with similar complete medium, CCCM provides a better condition for in vitro germ cell differentiation from hESCs. Moreover, a comparison between the 2 types of complete media showed that EB medium is more supportive than Dulbecco’s modified eagle’s medium +20% fetal bovin serum.
Conclusion: CCCM with EB medium as a complete medium provides a better condition for in vitro germ cell differentiation from hESCs.
This article has been extracted from Ph.D. Thesis. (Somayyeh Sadat Tahajjodi)
