Ovaries from 6 to 8 days old B6CBAF1 female pups genetically matching the receiving host were collected and transferred to Leibovitz L-15 media (Sigma-Aldrich, USA). The ovaries were dissected into 2–4 pieces and transferred in the maintenance media (α-MEM, Gibco, USA) in the CO2 incubator for further manipulation. Similarly, ovaries from 6 to 8 days old BALB/c were used as donor ovarian tissue for allogeneic implants.
Leibovitz l 15 media
Manufactured by Merck Group
Sourced in United States
Leibovitz L-15 medium is a cell culture medium developed by John Leibovitz. It is designed to support the growth of various cell types, including adherent and suspension cultures, in an atmosphere of 0% CO2.
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Lab products found in correlation
α mem, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Ethylene glycol, by Merck Group (1 mentions)
Iscove s modified dulbecco s media, by Merck Group (1 mentions)
Mda mb 231, by Merck Group (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Eclipse te200 s microscope, by Nikon (1 mentions)
Tryptose phosphate, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Cell strainer, by Merck Group (1 mentions)
Collagenase, by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Merck Group (1 mentions)
Percoll, by Merck Group (1 mentions)
11 protocols using leibovitz l 15 media
1
Prepubertal Ovarian Tissue Transfer
2
Isolation of Ovarian Cortical Tissue
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The use of human tissue was approved by the Health Research Authority South Central—Oxford B Research Ethics Committee (REC reference 14/SC/0041). Fresh whole ovaries (n = 3 patients) and frozen ovarian cortical tissue (n = 2 patients) were obtained from the Oxford Cell and Tissue Biobank (OCTB) (Table I ); OCTB obtained consent from the patients to donate this tissue for research. None of the patients had previously received chemotherapy or radiotherapy. The ovaries/ovarian tissue were removed as part of surgical procedures and donated for research.
The fresh ovaries were transported and dissected in cold (4°C) Leibovitz L-15 media (Sigma, Gillingham, UK, L5520) to isolate the ovarian cortex. The frozen cortical tissue was thawed in solutions of decreasing concentrations of ethylene glycol (1.0, 0.5, and 0 M) (Sigma, 324558), 0.1 M sucrose (Sigma, S7903), and 3 mg/mL human serum albumin (HSA; Sigma, A1653) for 5 min each at room temperature, using a rocking motion. All cortical tissue was further dissected in fresh L-15 into pieces approximately ≤1 mm3. Processing time between tissue collection from the OCTB and fixation was approximately 1 h.
The fresh ovaries were transported and dissected in cold (4°C) Leibovitz L-15 media (Sigma, Gillingham, UK, L5520) to isolate the ovarian cortex. The frozen cortical tissue was thawed in solutions of decreasing concentrations of ethylene glycol (1.0, 0.5, and 0 M) (Sigma, 324558), 0.1 M sucrose (Sigma, S7903), and 3 mg/mL human serum albumin (HSA; Sigma, A1653) for 5 min each at room temperature, using a rocking motion. All cortical tissue was further dissected in fresh L-15 into pieces approximately ≤1 mm3. Processing time between tissue collection from the OCTB and fixation was approximately 1 h.
3
Ovarian Tissue Isolation and Maintenance
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Donor ovaries were collected from 6–8 days old BALB/c mice. The collected ovaries were transferred to Leibovitz L-15 media (Sigma-Aldrich, St. Louis, USA) and dissected open. The ovarian tissue was then transferred into maintenance media (α-MEM; Gibco, Langley, USA), kept at 37 °C and 5% CO2 until encapsulated.
4
Culturing Cancer and MSC Cell Lines
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Cancer cell lines MDA-MB-231, MCF-7 and SW837 were obtained from the Russian cell culture collection (Russian Branch of the ETCS, Russia, St. Petersburg). MSC cells were kindly gifted by Dr. Matveeva [3] (link). MCF-7 and SW837 cells were cultivated in Iscove's modified Dulbecco's media (Sigma) with 10% FBS (Gibco BRL Co., Gaithersburg, MD), 2 mM L-glutamine (Sigma), 250 mg/mL amphotericin B and 100 U/mL penicillin/streptomycin (GIBCO BRL Co., Gaithersburg, MD). MDA-MB-231 cells were cultivated in Leibovitz (L15) media (Sigma) supplemented with 10% FBS, 2 mM L-glutamine, 250 mg/ml amphotericin B and 100 U/mL penicillin/streptomycin. Cells were grown in a humidified atmosphere of 5% CO2 in air at 37°C and were passaged with 0.05% trypsin-EDTA every 3–4 days.
5
Examination of Sexual Phenotypes in Mutant Fish
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To examine sexual phenotypes of sycp2+/- or spo11+/- mutant fish, offspring were obtained from mass mating of heterozygote fish. Offspring fish were dissected at 8 to 9-week old, and the morphology of the gonads was examined with a Nikon ECLIPSE TE200-S microscope. To facilitate the observation of gonad morphology, dissected gonads were placed on a slide with a drop of Leibovitz L-15 media (Sigma) and flattened with a coverslip. Gonads were classified into either ovaries with oocytes or testes with lobule structures and/or with spermatocytes, spermatids, or sperm cells (S14 Fig ). Fin clips of offspring fish were used for genotyping.
6
Ovarian Tissue Isolation and Maintenance
7
Isolation and Dissection of Murine Ovaries
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Ovaries from 6 to 8 days old BALB/c mice were collected and transferred to Leibovitz L-15 media (Sigma-Aldrich, USA). The ovaries were dissected into 2–4 pieces and transferred in the maintenance media (α-MEM, Gibco, USA) and placed in the CO2 incubator for further manipulation.
8
Aedes albopictus Cell Culture Protocol
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The
Aedes albopictus cell C636, kindly provided by Dr. Amílcar Tanuri from Universidade Federal do Rio de Janeiro, was cultivated in Leibovitz L-15 media (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich), 0.26% tryptose phosphate (Sigma-Aldrich), 100 units/mL of Penicillin and 100 μg/mL of Streptomycin (Sigma-Aldrich), at 28°C
13 (link).
Aedes albopictus cell C636, kindly provided by Dr. Amílcar Tanuri from Universidade Federal do Rio de Janeiro, was cultivated in Leibovitz L-15 media (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich), 0.26% tryptose phosphate (Sigma-Aldrich), 100 units/mL of Penicillin and 100 μg/mL of Streptomycin (Sigma-Aldrich), at 28°C
13 (link).
9
Ovarian Tissue Encapsulation in PEG Hydrogels
10
Isolation and Characterization of Leukocytes from Fish Organs
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The organs (CF, HK and SP) were isolated from five fish from each group and used to prepare single cell suspensions. HK and SP tissues were disaggregated in Dulbecco’s phosphate-buffered saline (DPBS; Sigma-Aldrich, Vienna, Austria) 2% NCS (FACS buffer, FB) in a Corning® cell strainer and then carefully layered onto 3 ml of Percoll (Sigma-Aldrich) in a 15 ml conical centrifuge tube, then centrifuged (with brake level 4) at 510×g for 30 min at 4 °C. The leukocytes were collected from the Percoll interface, washed once with FB and counted in a Neubauer chamber. Caudal fins were washed with 5 ml of DPBS containing 1 mM EDTA and 1 mM β-mercaptoethanol for 20 min in a shaking agitator at 5 °C. Then, the samples were washed 3 times with DPBS. The pre-treated fins were placed in new Petri plates and cut into small pieces, adding 0.15 mg/ml collagenase (Sigma-Aldrich) in Leibovitz L-15 media (Sigma-Aldrich). The digestion was performed for 30 min at room temperature with soft agitation. The supernatant was recovered and the pieces washed with FB and passed through a cell strainer. Samples were centrifuged at 510×g at 4 °C for 6 min; the cells were resuspended in 1 ml of FB and counted.
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