Cryopreservation Of Adipose Tissue Research Articles

Good Manufacturing Practice-Compliant Cryopreserved and Thawed Native Adipose Tissue Ready for Fat Grafting.

Background: As adipose tissue-derived mesenchymal stem cells are becoming the tool of choice for many clinical applications; standardized cryopreservation protocols are necessary to deliver high-quality samples. For this purpose, the cryopreservation and thawing of native adipose tissue under GMP conditions could represent an extremely useful and powerful tool for the direct reinfusion of the tissue, and consequently, of its stromal vascular fraction. Methods: In this study, 19 samples of adipose tissue were cryopreserved and characterized before and after storage in liquid nitrogen vapors. Of these 19 samples, 14 were processed in research and 5 in a GMP-compliant environment. Storage with and without cryopreservation medium was also evaluated. After one week to three months of storage, samples were thawed, washed, enzymatically digested, and characterized with flow cytometry. Results: The results show that there is a loss of nearly 50% of total nucleated cells during the cryopreservation/thawing process. Non-GMP and GMP samples are comparable for all parameters analyzed. This study also allowed us to exclude the cryopreservation of adipose tissue without any cryopreservation medium. Conclusions: The data shown in this work are consistent with the idea that native adipose tissue, if properly processed and controlled, could be a useful source of cells for regenerative medicine, keeping in mind that there is a clear difference in the quality between fresh and thawed samples.

The effect of glycerol as a cryoprotective agent in the cryopreservation of adipose tissue

BackgroundLong-term preservation of adipose tissue is crucial for clinical applications. Researchers should consider both efficiency and biosafety when choosing a cryoprotective agent (CPA) for adipose tissue preservation. Glycerol has been applied as a nontoxic CPA for multiple tissues but not adipose tissue. We aimed to evaluate the efficacy of glycerol as a CPA for adipose tissue cryopreservation.MethodsFresh human adipose tissues were obtained from patients who underwent liposuction and divided into 1 mL samples. Each sample was randomly mixed with 1 mL of CPA: 60–100% glycerol, 0.25 mol/L trehalose or DMSO + FBS and cryopreserved in − 196 °C liquid nitrogen for one month. After thawing and elution, the tissues were immediately evaluated for activity and structural integrity in vitro. Then, 0.2 mL of each sample was transplanted subdermally to the nude mouse dorsum and harvested after one month for histological examination to assess the effect of the cryopreserved fat in transplantation.ResultsAfter cryopreservation, the samples treated with DMSO + FBS, trehalose, 60% and 70% glycerol had a more integrated structure than the samples in other groups. Tissues preserved with 70% glycerol had the highest G3PDH activity of 24.41 ± 0.70, comparable to 24.76 ± 0.48 in fresh tissue (p > 0.05). Adipose-derived stem cells (ASC) viability, proliferation and differentiation capability were also better preserved in 70% glycerol group. In vivo analysis showed that tissue preserved with 70% glycerol had a retention rate of 52.37 ± 7.53%, significantly higher than other groups. Histological observation demonstrated better structural integrity and viability in 70% glycerol group. Compared to the DMSO + FBS and trehalose groups, the glycerol groups showed lower tissue inflammation.ConclusionGlycerol (70%) is efficient in adipose tissue cryopreservation. Glycerol-based CPAs, which are nontoxic and show biosafety, are a promising solution for clinical tissue cryopreservation.

Cryopreservation of adipose tissue with and without cryoprotective agent addition for breast lipofilling: A cytological and histological study

In the second reconstructive phase of the breast after mastectomy, lipofilling is often necessary. Currently, lipofilling occurs immediately after autologous adipose tissue harvesting procedure, but most of the patients, usually, require multiple sessions to obtain a satisfactory result. Therefore, the need of repeated surgical harvesting outputs implies high risk of patients’ morbidity and discomfort as well as increasing medical time and costs. The aim of our pilot study was to find out a feasible method to cryopreserve adipose tissue, in order to avoid reiterated liposuctions.Lipoaspirates samples have been harvested from 10 women and preserved by three methods: (1) the first one, using 10% Me2SO and 20% human albumin from human plasma as cryoprotective agents; (2) the second one, adding 5% Me2SO as cryoprotective agent; 3) the last one, without any cryoprotective agent. Fresh and cryopreserved fat samples, obtained through the aforementioned processes, have been analyzed ex vivo. The efficiency of the cryopreservation methods used was determined by adipocyte viability and the expression of adipocytes surface markers.Lipoaspirates stored at −196 °C for 3 months, after thawing, retained comparable adipocyte viability and histology to fresh tissue and no significant differences were found between the three methods used. Although the current results, differences between the methodologies in terms of viability may not become evident until breast lipofilling using frozen-thawed cryopreserved tissue.

Human Adipose Tissue Cryopreservation: Impact of Different Calf Serum Concentrations on Tissue Viability.

Adipose tissue, which is harvested in large quantities during liposuction, has no blood supply and becomes necrotic within a few hours, if not immediately transplanted. Cryopreservation of adipose tissue allows these samples to be stored and used in diverse fundamental experiments, especially in fat-grafting animal tests that could provide a theoretical basis for clinical applications. Traditionally, fetal bovine serum (FBS) has been added as a cryoprotectant (CPA) to maintain the maximum viability of different tissues after freezing and thawing. Calf serum (CS) comes from the same species as FBS but is more economical compared with FBS-containing medium. The optimal concentration of CS in CPA for banking adipose tissue has not been studied. Here, we studied the cell survival rate, cell viability, tissue structural integrity, number of adipose-derived stem cells and blood vessels, and survival after transplantation into nude mice via ultrastructural evaluation of adipose tissue cryopreserved for 6 months in condition A (60% CS, 15% dimethyl sulfoxide [DMSO], 25% Dulbecco's modified Eagle's medium [DMEM]) and condition B (30% CS, 15% DMSO, 55% DMEM). Our results indicate that CS in addition to CPA results in adequate preservation of adipose tissue, especially when a higher concentration of CS (60%) is used in the CPA.

An Update on Cryopreservation of Adipose Tissue.

Currently, fat transplantation occurs immediately after harvesting procedures. Because low rates of fat graft take are well reported in the literature, many patients require multiple surgical procedures for fat graft harvest. These subsequent procedures lead to increased cost, donor-site morbidity, and patient discomfort in the long term. The ability to preserve our patients' own adipose aspirate would allow us to counteract these shortcomings and ultimately improve the clinical outcome after fat grafting. Unfortunately, there is no optimal and practical adipose tissue cryopreservation protocol for use by the plastic surgeon at the present time. Because of this dilemma, the senior author (L.L.Q.P.) has investigated this concept in an effort to create a protocol that is both technically sound and clinically achievable to allow for the long-term preservation of adipose tissue. In this article, the authors aim to outline this effort, review current clinical applications that have been reported in the literature, and detail exciting future perspectives in the use of preserved lipoaspirates for repeated fat grafting procedures or in the form of cell-based therapy engineered for reconstructive endeavors for their patients.

Long-Term Biobanking of Intact Tissue from Lipoaspirate.

Autologous fat grafting has now been extensively and successfully performed for more than two decades. Although most adipose grafts and adipose-derived MSC therapies are done with fresh tissue, cryopreservation of tissue allows for much greater flexibility of use. Over the course of five years, 194 cryopreserved adipose samples were thawed and then returned to the collecting physician for subsequent autologous applications. Samples were stored with a mean cryogenic storage time of 9.5 months, with some samples being stored as long as 44 months. The volumes of tissue stored varied from 12 cc to as large as 960 cc. Upon thawing, the volume of recovered whole adipose tissue averaged 67% of the original amount stored for all samples, while the samples that were stored for longer than one year averaged 71%. Recovery was not found to be a function of length of time in cryopreservation. No significant relationship was found between tissue recovery and patient age. While an average recovery of 67% of volume frozen indicates that the use of banked and thawed tissue requires a larger amount of sample to be taken from the patient initially, an experienced clinician easily accomplishes this requirement. As cryopreservation of adipose tissue becomes more commonplace, physicians will find it helpful to know the amount and quality of tissue that will be available after thawing procedures.

Isolation of Adipose-Derived Stem/Stromal Cells from Cryopreserved Fat Tissue and Transplantation into Rats with Spinal Cord Injury.

Adipose tissue contains multipotent cells known as adipose-derived stem/stromal cells (ASCs), which have therapeutic potential for various diseases. Although the demand for adipose tissue for research use remains high, no adipose tissue bank exists. In this study, we attempted to isolate ASCs from cryopreserved adipose tissue with the aim of developing a banking system. ASCs were isolated from fresh and cryopreserved adipose tissue of rats and compared for proliferation (doubling time), differentiation capability (adipocytes), and cytokine (hepatocyte growth factor and vascular endothelial growth factor) secretion. Finally, ASCs (2.5 × 106) were intravenously infused into rats with spinal cord injury, after which hindlimb motor function was evaluated. Isolation and culture of ASCs from cryopreserved adipose tissue were possible, and their characteristics were not significantly different from those of fresh tissue. Transplantation of ASCs derived from cryopreserved tissue significantly promoted restoration of hindlimb movement function in injured model rats. These results indicate that cryopreservation of adipose tissue may be an option for clinical application.

Isolation and cryopreservation of adipose tissue derived stem cells from different adipose tissues in obese patients

Abstract Introduction: Adult stem cells (ASCs) are a population of tissue-resident cells that have the capacity for self-renewal and differentiation into different cell types with potential for cell therapies. New approaches for ASCs isolation, including many tissue sources and new protocols that are more effective and less expensive are under investigation. Thus this work aim is to isolate, maintain in cell culture and evaluate cryopreservation protocols for adipose derived stem cells (ADSCs) from different tissues such as subcutaneous adipose tissue, visceral mesenteric and omental visceral taken from the same individual. Material and Methods: The techniques of mechanical and enzymatic dissociation were used, in order to investigate the most appropriated method to ADSCs isolation. Dimethylsulfoxide (DMSO) and dimethylformamide (DMF) in different concentrations were tested as cryoprotectors in 24, 48 and 72 hours thawing protocols. The samples were collected from obese patients with associated diseases undergoing bariatric surgery, between 30 to 45 years old. Results: Among 10 collected samples it was possible to measure cell viability from 4 patients. The higher cell rate was obtained from the visceral tissue of omentum. Conclusion: DMSO was the more efficient cryopreservant for this condition. This adipose tissue source could be explored for ADSCs isolation and future clinical investigations.

Patient use of autologous cryopreserved intact adipose tissue from lipoaspirate

Autologous fat grafting has now been extensively and successfully performed for more than two decades. Although most adipose grafts and adipose-derived MSC therapies are done with fresh tissue, cryopreservation of tissue allows much greater flexibility of use. Over the course of five years, 194 cryopreserved adipose samples were thawed and returned to the collecting physician for subsequent autologous applications. Samples were stored with a mean cryogenic storage time of 9.5 months with some samples being stored as long as 44 months. The volumes of tissue stored varied from 12 cc to as large as 960 cc. Upon thaw the volume of recovered whole adipose tissue averaged 67% of the original amount stored. The recovery ranged from a low of 21% (n = 1) to a high of 100% (n = 1). Recovery was not found to be a function of collection volume, amount of tissue thawed, or length of time in cryopreservation. No association was found between tissue recovery and patient age. Viability of thawed cells remained high with a mean value of 91%. While an average recovery of 67% of volume frozen indicates that the use of banked and thawed tissue requires a larger amount of sample be taken from the patient initially, this requirement is easily accomplished by an experienced clinician. As cryopreservation of adipose tissue becomes more commonplace physicians will find it helpful to know the amount and quality of tissue that will be available after thawing procedures.

Adipose Derived-Mesenchymal Stem Cells Viability and Differentiating Features for Orthopaedic Reparative Applications: Banking of Adipose Tissue.

Osteoarthritis is characterized by loss of articular cartilage also due to reduced chondrogenic activity of mesenchymal stem cells (MSCs) from patients. Adipose tissue is an attractive source of MSCs (ATD-MSCs), representing an effective tool for reparative medicine, particularly for treatment of osteoarthritis, due to their chondrogenic and osteogenic differentiation capability. The treatment of symptomatic knee arthritis with ATD-MSCs proved effective with a single infusion, but multiple infusions could be also more efficacious. Here we studied some crucial aspects of adipose tissue banking procedures, evaluating ATD-MSCs viability, and differentiation capability after cryopreservation, to guarantee the quality of the tissue for multiple infusions. We reported that the presence of local anesthetic during lipoaspiration negatively affects cell viability of cryopreserved adipose tissue and cell growth of ATD-MSCs in culture. We observed that DMSO guarantees a faster growth of ATD-MSCs in culture than trehalose. At last, ATD-MSCs derived from fresh and cryopreserved samples at −80°C and −196°C showed viability and differentiation ability comparable to fresh samples. These data indicate that cryopreservation of adipose tissue at −80°C and −196°C is equivalent and preserves the content of ATD-MSCs in Stromal Vascular Fraction (SVF), guaranteeing the differentiation ability of ATD-MSCs.

Successful isolation of viable adipose-derived stem cells from human adipose tissue subject to long-term cryopreservation: positive implications for adult stem cell-based therapeutics in patients of advanced age.

We examined cell isolation, viability, and growth in adipose-derived stem cells harvested from whole adipose tissue subject to different cryopreservation lengths (2–1159 days) from patients of varying ages (26–62 years). Subcutaneous abdominal adipose tissue was excised during abdominoplasties and was cryopreserved. The viability and number of adipose-derived stem cells isolated were measured after initial isolation and after 9, 18, and 28 days of growth. Data were analyzed with respect to cryopreservation duration and patient age. Significantly more viable cells were initially isolated from tissue cryopreserved <1 year than from tissue cryopreserved >2 years, irrespective of patient age. However, this difference did not persist with continued growth and there were no significant differences in cell viability or growth at subsequent time points with respect to cryopreservation duration or patient age. Mesenchymal stem cell markers were maintained in all cohorts tested throughout the duration of the study. Consequently, longer cryopreservation negatively impacts initial live adipose-derived stem cell isolation; however, this effect is neutralized with continued cell growth. Patient age does not significantly impact stem cell isolation, viability, or growth. Cryopreservation of adipose tissue is an effective long-term banking method for isolation of adipose-derived stem cells in patients of varying ages.

Cryopreservation of Adipose Tissue for Cosmetic Treatment of HIV Related Facial Lipoatrophy is Underused

Few infectious diseases have the social stigma or the emotional power that is often associated with HIV. The psychological impact of any disease can be its own difficult, and occasionally insurmountable, hurdle that has very little to do with science or medicine. However, HIV infection seems to have taken on the mantle of a disease with the greatest overall social impact. Over more than three decades of research vast new understanding of the infection and disease progression has taken place. Current states of knowledge have allowed the development of drug cocktails (HAART therapy) that keep a large fraction of morbidity and mortality at bay. But as HIV+ patients live longer and better lives, they must still contend with prejudice and stigma that sometimes associates with disease status. This is often due to the simple fact that HIV+ people look like HIV+ people. As the disease progresses, elements of HIV associated lipodystrophy can change the individuals appearance. Banking of adipose tissue early after diagnosis for subsequent autologous use years later can offer patients a safe and affordable surgical option for reversing the changes in facial appearance and social stigma that associate with HIV status.

Adipogenic Differentiation of Human Adipose Tissue–Derived Stem Cells Obtained from Cryopreserved Adipose Aspirates

Although frozen adipose tissue is frequently used for soft tissue augmentation, the viability of frozen fat remains a controversy. The cryopreservation of adipose tissue is important for the future use of adipose-derived stem cells (ASCs) and adipocytes. To determine whether optimal cryopreservation techniques with regard to the addition of cryopreservative agents and preservation temperature is essential for the long-term storage of adipose tissue and whether ASCs from cryopreserved adipose aspirates are reliable for use in adipogenic differentiation. Adipose tissue was frozen directly or with cryoprotectant at -20 degrees C or -80 degrees C for 1 year. The viability of adipose aspirates and the differentiation of ASCs isolated from adipose tissue were evaluated. The viability of adipose aspirates frozen with dimethyl sulfoxide at -80 degrees C was approximately 87% after 2 months of storage. Moreover, ASCs from adipose tissue stored with cryoprotectant survived successfully for 1 year and differentiated into adipocytes, although ASCs were not detected in the directly frozen adipose tissue. Adipose tissue cryopreserved with cryoprotectant and stored at optimal temperature might prove to be a reliable source of human ASCs and adipocytes.

A Novel Agent for Improved Cryopreservation of Adipose Tissue

Background: Improved cryopreservation of fat would significantly enhance reconstruction with fat grafts by providing for multiple treatments without additional harvesting. Currently, cryopreservation is limited by the toxicity of standard agents such as DMSO. In our study of adipocyte resuscitation using a tri-block copolymer (P188) we have been able to demonstrate a significant improvement in graft preservation. We hypothesized that a similar strategy may be utilized to protect cryopreserved fat as well.