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PubMed2026

Beyond survival: Testicular tissue cryopreservation for fertility preservation during childhood cancer, experiences from a single center.

INTRODUCTION: Approximately 30% of all male childhood cancer survivors receive gonadotoxic therapy increasing their risk for infertility. The only current option for potential fertility preservation in prepubertal males is through testicular tissue cryopreservation (TTC), available through clinical trials. Our aim was to evaluate our experience and identify barriers to enrollment in the TTC study. METHODS: A retrospective review was conducted to describe a single center experience with TTC, including an assessment of factors impacting the decision to enroll or not enroll in the TTC trial. RESULTS: A total of 61 patients were found to be eligible for enrollment in the TTC trial, with 23 patients (34%) choosing to enroll and undergo testicular tissue cryopreservation including 19 (83%) oncology patients. In patients who underwent TTC there were no unexpected delays in starting disease directed therapy. For families that declined enrollment, many did not see fertility preservation as a priority at the time of counseling. (n = 17, 44%). Other barriers included concerns about post-operative complications and a feeling of being overwhelmed at the time of counseling. DISCUSSION: Despite being the only preservation option for pre-pubertal boys facing risk of infertility, TTC enrollment was not pursued by a majority of eligible patients and families. Identifying potential barriers or misconceptions, particularly in regard to post operative complication risks, offer opportunities for improved counseling and education. Early introduction of the Fertility Navigation team allows for procedural coordination success and access for patients that are interested. Success is not measured in how many patients proceed with TTC, rather that all patients have the opportunity for counseling and discussion. CONCLUSION: TTC can be offered and completed safely without delays in disease-directed treatment timelines. A Fertility Navigation Program allows for equal opportunity for education and enrollment on TTC protocol which should be the goal for pediatric institutions, though is not pursued by many patients. A majority of those who declined the study did not see fertility as a priority at the time of treatment while others were concerned regarding peri-operative risks.

Humans↗
PubMed2018

Chromatin architecture changes and DNA replication fork collapse are critical features in cryopreserved cells that are differentially controlled by cryoprotectants.

In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.

Cell Survival↗
PubMed1978

Penetrating keratoplasty with fresh and cryopreserved corneas. Donor endothelial cell survival in primates.

Eight monkeys received bilateral corneal transplants, one fresh and one cryopreserved, from eight other healthy monkeys of sex opposite to that of the recipients. Three of the animals maintained clear grafts in both eyes, and the sex chromatin patterns of the corneal endothelial cells in these transplants were studied at one, two, and three years after keratoplasty. Definite survival of donor endothelial cells of their progeny was observed in each graft, and the percentage of cells of donor origin was similar for both fresh and cryopreserved grafts at each timer period.

Animals↗
PubMed1976

Cryopreservation of human lymphocyte function as measured by in vitro assays.

Methods are described by which cryopreserved cells can be utilized in a number of in vitro assays. On a per cell basis, nearly total recovery of function can be demonstrated for lymphocyte transformation (mitogens, antigens and MLC) and rosette-forming cells. Excellent recovery of mononuclear cell production of leukocyte migration inhibitory factor was also observed. Highly reproducible activity was recovered on a per lymphocyte basis in lymphocyte cytotoxicity but with a definite decrement in the percentage recovery. Both for longitudinal studies of immune function and for standardization of these assays in one or more laboratories such cryopreserved cells are ofimmense value and should be widely utilized.

Blood Specimen Collection↗
PubMedDate not supplied

Cryopreservation of human lymphoid cells from various tissues.

Normal and pathological lymphoid cells, collected from different sources, were cryopreserved using a programmed freezing procedure. With this cryopreservation technique, the percentage of T and B cell surface markers and the proliferative response to mitogens were not influenced by 21 and 35 days of storage in liquid nitrogen. The recovery percentage of the lymphoid cells was satisfactory when fetal calf serum was added, as a protein source, to the medium during freezing and thawing phases, while a very low percentage of cells was recovered if the fetal calf serum was omitted.

Antigens, Surface↗
PubMed1976

Cryopreserved endothelial cell survival in vivo.

We observed the endothelium of a cryopreserved penetrating corneal transplant with the clinical specular microscope at intervals from one day to two months after keratoplasty. An intact endothelial mosaic was observed at each examination, documenting the survival of cryopreserved endothelial cells after penetrating keratoplasty in humans.

Aged↗
PubMed1979

In vitro induction of cytotoxic effector cells against human neoplasms. I. Sensitization conditions and effect of cryopreservation on the induction and expression of cytotoxic responses to allogeneic leukemia cells.

Peripheral blood lymphocytes (PBL) from normal human donors were sensitized in vitro against allogeneic human acute myelocytic leukemia (AML) cells by means of an unidirectional mixed lymphocyte-tumor cell culture (MLTC) technique. The cytotoxic responsiveness of the sensitized lymphocytes, as determined in vitro by the 51Cr-release assay, varied among individual lymphocyte donors and was greatly dependent on the sensitization culture conditions. Induction of cytotoxic effector cells was augmented appreciably by adding to the cultures minute amounts of the immunopotentiating agent MER-BCG. Responding lymphocytes and stimulating leukemia cells cryopreserved for several weeks in liquid nitrogen were as effective as fresh cells in generating effector lymphocytes; the cytotoxic capacity of already sensitized lymphocytes was fully retained by cryopreservation. The implications of these findings for possible clinical employment of in vitro sensitized lymphocytes in adoptive immunotherapy of cancer are discussed.

Adult↗
PubMed2026

Cryopreservable dopaminergic progenitors derived from human iPSCs with accelerated loss of pluripotency and early functional restoration in Parkinsonian rats.

Midbrain dopaminergic progenitors (mDAp) derived from human pluripotent stem cells have demonstrated promising safety and efficacy in Phase I clinical transplantation trials for Parkinson's disease (PD). To further improve the translational potential of this approach, strategies that accelerate the loss of pluripotency, increase the yield of in vitro mDA progenitors, and promote post-transplantation neurite outgrowth may be beneficial. Here, we developed an optimized protocol building upon our previously established neural induction method, with further refinements, to efficiently convert induced pluripotent stem cells (iPSCs) into mDA neurons. Additionally, we applied n-butylidenephthalide, which selectively reduced pluripotency-associated gene expression and enhanced neurite outgrowth during differentiation. With these improved techniques, 71% of iPSCs differentiated into mDA neurons, showing burst dopamine secretion and phasic electrophysiological activities with external stimuli. To evaluate the safety, cryopreserved mDAp were transplanted into immunodeficient mice. No teratoma or neural tumor was observed within 24 weeks post-implantation. In 6-OHDA PD rats, mDAp survived and differentiated into mDA neurons in the host striatum within eight weeks post-transplantation, leading to significant functional recovery. The current differentiation process, therefore, enables the generation of cryopreservable, off-the-shelf mDAp, with accelerated loss of the pluripotency marker OCT4 in vitro and early functional recovery following transplantation.

Journal Article↗
PubMedDate not supplied

Cryopreservation of human lymphocytes: a brief review and evaluation of an automated liquid nitrogen freezer.

Successful cryopreservation of human lymphocytes has been previously described. Cryopreserved lymphocytes are useful for a variety of in vitro immunologic studies. This study was performed to determine the applicability and/or advantages of using a programmable freezing system, and compares glycerol versus dimethyl sulfoxide (DMSO) at varying concentrations on post-thaw viability, E-rosetting and immunoglobulin fluorescence. Prefreeze T and B lymphocyte percentages were determined. Cells were then frozen in varying concentrations of glycerol and DMSO. Optimum cryoprotectant type and concentration was determined. Lymphocytes from seven individuals were frozen by the batch method in a mechanical freezer and with the automated liquid nitrogen injection system. Data on post-thaw T and B percentages and viability revealed 10% DMSO and liquid nitrogen control freezing method at 1 C/minute as the best conditions for lymphocyte preservation as reflected by post-thaw in vitro testing.

Blood Preservation↗
PubMed1979

The survival of cryopreserved human bone marrow stem cells.

Two methods for cryopreservation of bone marrow stem cells were compared using bone marrow obtained from 36 patients. Included in this group were 21 persons with the diagnosis of leukaemia including 14 either with acute myeloid or lymphoblastic leukaemia in remission following intensive remission induction chemotherapy. After freeze-preservation and reconstitution, all marrow samples were tested for nucleated cell (NC) recovery and grown on agar to assess colony forming units (CFUC) and cluster forming units in culture (CluFUc). A slow dilution reconstitution method using freezing media containing AB negative plasma resulted in recovery of 85% of the CFUc activity of fresh marrow. This result was significantly better than the 47% CFUc recovery obtained when freezing media without plasma and a rapid dilution reconstitution technique were used. NC recoveries following slow dilution (51%) and rapid dilution (44%) were not significantly different. CluFUc were disproportionately reduced compared with CFUc although yielding similar results with both methods (26% and 32%). No correlation was found for either method between CFUc and NC recovery or between CFUc and CluFUc recovery in cryopreserved bone marrow.

Adolescent↗
PubMed1979

The cryopreservation and in vitro cultivation of larval Onchocerca volvulus.

A system developed in our laboratory for the in vitro cultivation of larval Onchocerca volvulus is being employed in a series of morphogenetic, physiologic, chemotherapeutic and immunologic investigations of this parasite. Because of the need for a large and readily available supply of living worms for this work, cryogenic techniques are being used for the long-term preservation of larval parasites collected in various endemic areas of Guatemala, C.A. To date, microfilariae have survived frozen storage in human cutaneous tissues (excised nodules and skin snips) for as long as 504 days, and viable larvae, in all stages of development have been recovered from the black fly vectors (Simulium ochraceum and S. metallicum) kept frozen for 396 days. That cryopreservation does not appear to affect these parasites adversely is indicated by the fact that microfilariae derived from frozen tissues do not differ from those obtained from fresh tissues on the basis of: 1) numbers and vigor of emergent microfilariae; 2) survival and morphogenesis of microfilariae during cultivation in vitro for 24 days; 3) glucose utilization during 72 hours of incubation; and 4) their incorporation of 3H-amino acids as determined after 18 hours of incubation. Details of methodology for cryopreservation and in vitro cultivation, together with resultant data, are presented herein.

Animals↗
PubMed1976

The processing of human bone marrow for cryopreservation and reinfusion.

Bone marrow was aspirated from 22 patients with solid tumors. A median of 1.2 x 10(10) net bone marrow cells were obtained during a 1 hour 45 minute operative procedure. Three methods were used to concentrate the bone marrow for cryopreservation. The greatest concentration of mononuclear cells was obtained by the Haemonetics pheresis procedure. Bone marrow was cryopreserved in 10 % DMSO in volumes ranging from 150 to 700 ml. The thawed marrow was further processed to remove both DMSO and free hemoglobin prior to reinfusion. Cell clumping was observed in the first 6 of the 10 marrows thawed. Acidification of the thawed marrow to pH 6.65 was found to retard cell clumping. The immediate recovery of cells frozen was 66 % with a viability of 50 %. The mean number of viable mononuclear bone marrow cells per kilogram available for reinfusion was therefore 5.5 x 10(7).

Bone Marrow↗
PubMed1979

Lack of genetic damage in mammalian cells after cryopreservation at -196 degrees C.

Mammalian tissue culture cells when cryopreserved according to standard procedures do not show gross chromosomal changes as evidenced by the formation of micronuclei. Cryoprotective agents used in this study included dimethyl sulfoxide (DMSO), glycerol and polyvinylpyrollidone (PVP) in combination with serum and using freezing and thawing rates normally employed in cryopreservation studies. In preliminary experiments, point mutations as evidenced by oubain resistance were not observed. These results are discussed in relation to previous studies indicating genetic stability after freezing and thawing.

Animals↗
PubMed1979

Cryopreservation of bovine mononuclear leukocytes.

A technique of cryopreservation of bovine mononuclear leukocytes for use in lymphocyte stimulation tests and cell identification studies has been developed. Dimethyl sulfoxide was used as the cryopreservation agent. Cells were frozen to --40 C at a controlled rate of --1 C/minute and then to --80 C at a rate of --4 C/minute, and were subsequently stored in liquid nitrogen (--109 C). The cells were than recovered by rapid thawing in a 37-C water bath, diluted with tissue culture media, washed, and used for lymphocyte stimulation and cell identification tests. The magnitude of the lymphocyte blastogenic responses of frozen and thawed mononuclear leukocytes was similar to that seen with freshly collected cells. Additionally, percentages of T cells, B cells, and monocytes were similar between frozen and thawed cells and freshly collected cells.

Animals↗
PubMed1979

Accelerated hemopoietic recovery following the infusion of cryopreserved autologous bone marrow in humans.

High dose combination chemotherapy was given to 22 patients with malignant lymphoma resistant to conventional chemotherapy. This was followed in 12 patients by an infusion of their cryopreserved autologous bone marrow; 10 patients received chemotherapy alone and serve as controls. Patients receiving marrow recovered leukocyte, granulocyte, and platelet function significantly faster than controls demonstrating that cryopreserved autologous bone marrow infusions accelerate hemopoietic recovery. Four patients with Burkitt's lymphoma resistant to conventional chemotherapy appear cured of their disease following this single treatment with higher doses of chemotherapy.

Bone Marrow Transplantation↗
PubMed1979

Successful transfusion of platelets cryopreserved for more than 3 years.

To determine the duration of storage for cryopreserved platelets, 14 transfusions of random-donor, pooled platelets, stored in the vapor phase of liquid nitrogen for a mean period of 1157 days (range 1060-1240), were analyzed. Twelve of these transfusions were compared in a paired fashion with fresh, random-donor, pooled platelets given within a few days to the same thrombocytopenic recipients. Platelets had been frozen using 5% dimethylsulfoxide as a cryoprotective agent either at a controlled rate of -1 degrees C/min to -80 degrees C or by simply placing them in the vapor phase (-120 degrees C) of a liquid nitrogen freezer. The mean freeze-thaw loss for the 14 transfusions was 22%, and the mean corrected 1-hr increment in platelet count was 12,600/microliter. In the 12 paired observations, the mean corrected 1-hr increment for frozen platelets was 11,800/microliter and 25,900 for fresh platelets, giving a frozen/fresh recovery of 46%. Random donor platelets can be cryopreserved by these methods for greater than 3 yr with satisfactory post-transfusion increments. This suggests that a reservoir of frozen platelets, either random-donor for emergency transfusion or of known HLA-type for transfusion to alloimmunized patients, can be established and stored for at least 3 yr.

Blood Platelets↗
PubMed1977

Cryopreservation of the infective larvae of the common nematodes of ruminants.

Exsheathed infective larvae (L 3) of 19 species of nematodes were tested for infectivity in either sheep or cattle after they had been frozen in 0,9% NaCl solution, stored for a relatively short time in the gas phase of liquid nitrogen and subsequently thawed. In addition, 13 of these species were tested after similar storage for up to 18 months. In sheep, Haemonchus contortus, Ostertagia circumcincta, Trichostrongylus axei, Trichostrongylus colubriformis, Nematodirus spathiger and Oesophagostomum columbianum were viable after 2 years of cryopreservation, a mean of greater than 90% of the L 3 being alive when thawed after this period. Similar results were obtained with Chabertia ovina L 3 after 18 months and with Marshallagia marshalli, Trichostrongylus falculatus and Dictyocaulus filaria, after a short period of freezing. On the other hand, Gaigeria pachyscelis and Strongyloides papillosus survived freezing for up to 7 months but neither was viable at the end of this period, nor was exsheathed G. pachyscelis viable without freezing. Most of these infestations were established by inoculating the infective larvae into the abomasum and/or duodenum. M. marshalli, T. falculatus and C. ovina also proved infective after oral dosing. D. filaria, the only other species tested by this route, was not infective when dosed per os after thawing. The infective larvae of the bovine nematodes, Haemonchus placei, Ostertagia ostertagi, Nematodirus helvetianus, Oesophagostomum radiatum, Cooperia pectinata and Cooperia punctata survived freezing for a mean of 26 months, greater than 90% being alive on thawing, but infectivity was generally lower than with the same genera in sheep. Even when not frozen, exsheathed Bunostomum phlebotomum was non-infective. When Cooperia spp. after thawing were tested for infectivity by the oral route, more worms developed in one calf infested orally than in another infested by inoculation into the duodenum. Ova of H. contortus, M. marshalli, O. circumcincta, T. colubriformis, T. falculatus, N. spathiger, C. ovina, H. placei, O. ostertagi, Cooperia spp. and N. helvetianus were recovered from the faeces of animals infested with cryopreserved L 3. No ova of O. columbianum or O. radiatum were recovered from faeces, because differential larval counts were performed before they were patent. Nevertheless, gravid females were obtained post-mortem. Frozen L 3 of N. helvetianus were used to re-establish a pure strain in calves, 2,3 million ova being recovered from infestations with 10 670 L 3 frozen for 26 months. The infectivity of the progeny of frozen L 3 was tested with M. marshalli and C. ovina. In both instances infectivity was high and the worms which developed also produced ova, thus completing the cycle. This appears to be the first report of infective larvae of parasitic nematodes retaining their infectivity after being frozen in liquid nitrogen (gas phase) for longer than 2 years. This is also apparently the first time that M. marshalli T. colubriformis, T. falculatus, T. axei, N. spathiger, C...

Ancylostomatoidea↗
PubMed1978

Function of cryopreserved human effector cells in antibody-dependent, cell-mediated cytotoxicity.

Cells active in an ADCC model were studied for their ability to withstand cryopreservation. For this model, we used the [3H]proline microcytotoxicity assay with fibroblast target cells and a serum containing lymphocyte-dependent antibody reactive with the alloantigens on those target cells. Human blood effector cells active in this system can be successfully cryopreserved with minor dimunition in function with standard techniques and concentrations between 9 and 12% DMSO.

Antibody-Dependent Cell Cytotoxicity↗