Cryopreservation of collagen-based tissue equivalents. I. Effect of freezing in the absence of cryoprotective agents

The effect of freezing on the viability and mechanical properties of tissue-equivalents (TEs) was determined under a variety of cooling conditions, with the ultimate aim of optimizing the cryopreservation process. TEs (a class of bioartificial tissues) were prepared by incubating entrapped human foreskin fibroblasts in collagen gels for a period of 2 weeks. TEs were detached from the substrate and frozen in phosphate-buffered saline using a controlled rate freezer (CRF) at various cooling rates (0.5, 2, 5, 20, and 40 degrees C/min to -80 or -160 degrees C) or in a directional solidification stage (DSS) (5 degrees C/min to -80 degrees C) or slam frozen (>1000 degrees C/min). Viability of the fibroblasts in the TEs was assessed by ethidium homodimer and Hoechst assays immediately after thawing. Uniaxial tension experiments were also performed on an MTS (Eden Prairie, MN) Micro Bionix system to assess the postthaw mechanical properties of the frozen-thawed TEs. Cooling rates of either 2 or 5 degrees C/min using the CRF were optimal for preserving both immediate cell viability and mechanical properties of the TEs, postthaw. By 72 h postthaw, TEs frozen in the CRF at 5 degrees C/min to -80 degrees C showed a slight decrease in cell viability, with a significant increase in tangent modulus and ultimate tensile stress suggesting a cell-mediated recovery mechanism. Both the postthaw mechanical properties and cell viability are adversely affected by freezing to the lower end temperature of -160 degrees C. Mechanical properties are adversely affected by freezing in the DSS.     

Effects of Freezing and Cryopreservation on the Mechanical Properties of Arteries

Cryoplasty, a freezing therapy, is being used for the treatment of restenosis in peripheral arteries. In addition, cryo-preserved arteries are increasingly used in vascular grafts. While studies are being performed to establish the efficacy of such treatments, very little is known about the postcryosurgical or postcryo-preservation changes in mechanical properties of the arteries. Few studies have examined the effect of freezing in the absence of cryoprotective agents (CPAs), and the several studies done in the presence of CPAs have given mixed results. To examine this issue further, we froze pig femoral arteries in a controlled rate freezer, using an aluminum probe, both in the presence at (−80°C to 1°C/min) and absence (at −20°C for 2 or 5 mins) of CPA and Fetal bovine serum (FBS). Following freezing, artery samples were subjected to uniaxial tensile testing. The weights of the tissue were measured before and after freezing. Our results suggest that freezing does have an effect on stress-strain properties, particularly in the low stress region corresponding to physiological conditions. The mechanisms of this change in mechanical properties may include the loss of smooth muscle cell viability, damage to extra cellular matrix (ECM), bulk redistribution of water, or changes in alignment caused by ice crystal growth. In the case of samples frozen in the absence of CPA or FBS, the results indicated a drastic reduction in weight of the tissue suggesting the importance of bulk water redistribution as one underlying mechanism. To further examine potential mechanisms, we subjected cryopreserved vessels to the same uniaxial tests. The extent of changes in mechanical properties and bulk water redistribution was greatly attenuated; reinforcing that water movement might play a role in the changes observed with freezing.     

Polyvinylpyrrolidone (PVP) Mitigates the Damaging Effects of Intracellular Ice Formation in Adult Stem Cells

The objective of this work was to assess the effect of 10% (w/v) polyvinylpyrrolidone (PVP) on the pattern of intracellular ice formation (IIF) in human adipose tissue derived adult stem cells (ASCs) in the absence of serum and other cryoprotective agents (CPAs). The freezing experiments were carried out using a fluorescence microscope equipped with a Linkam™ cooling stage using two cooling protocols. Both the cooling protocols had a common cooling ramp: cells were cooled from 20 °C to −8 °C at 20 °C/min and then further cooled to −13 °C at 1 °C/min. At this point we employed either cooling protocol 1: the cells were cooled from −13 °C to −40 °C at a pre-determined cooling rate of 1, 5, 10, 20, or 40 °C/min and then thawed back to 20 °C at 20 °C/min; or cooling protocol 2: the cells were re-warmed from −13 °C to −5 °C at 20 °C/min and then re-cooled at a pre-determined rate of 1, 5, 10, 20, or 40 °C/min to −40 °C. Almost all (>95%) of the ASCs frozen in 1× PBS and protocol 1 exhibited IIF. However, almost none (<5%) of the ASCs frozen in 1× PBS and protocol 2 exhibited IIF. Similarly, almost all (>95%) of the ASCs frozen in 10% PVP in PBS and protocol 1 exhibited IIF. However, ~0, ~40, ~47, ~67, and ~100% of the ASCs exhibited IIF when frozen in 10% PVP in PBS and utilizing protocol 2 at a cooling rate of 1, 5, 10, 20, or 40 °C/min, respectively.     

The effects of storage conditions on viability of Clostridium difficile vegetative cells and spores and toxin activity in human faeces

AIMS:Clostridium difficile is a common nosocomial pathogen and as such diagnostic and research methods may necessitate storage of faecal specimens for long periods, followed by subsequent re-examination. This study investigated the effects of storage conditions upon the viability of this organism and its toxin. METHODS: Three genotypically distinct strains of C difficile (two clinical isolates including the UK epidemic strain, and an environmental isolate) were grown anaerobically at 37 degrees C for 72 hours in a pool of five faecal emulsions. Aliquots of each emulsion were stored at either -20 degrees C (frozen) or 4 degrees C (refrigerated). Emulsions were assayed for viable cells, spores, and cytotoxin titre before storage and at days 1, 3, 5, 7, 14, 28, and 56. An aliquot of each emulsion was also removed, assayed, and replaced in storage at each time point to investigate the effects of multiple freezing/refrigeration/thawing. RESULTS: Neither storage temperature nor multiple cycles of refrigeration/freezing and thawing adversely affected the viability of C difficile vegetative cells or spores. Single and multiple exposures of samples to 4 degrees C had little effect upon the C difficile toxin titre. Toxin titres of multiply frozen and thawed faeces became significantly lower than for refrigerated faeces (p < 0.01) by day 5 of the experiment in two of the three strains, and in all strains by day 28. Toxin titres of singly frozen faeces became significantly lower than for refrigerated faeces (p < 0.01) by day 56 of the experiment in two of the three strains. CONCLUSION: Storage temperature and multiple cycles of freezing (refrigeration)/thawing had minimal effects upon the viability of C difficile or its spores. Storage at 4 degrees C has no discernible effect on C difficile cytotoxin. However, storage at -20 degrees C has a detrimental effect upon C difficile cytotoxin, and multiple cycles of freezing and thawing may further adversely effect toxin titres.     

Cryogenic preservation of virus-infected cells used as immunofluorescent assay substrates

Cells infected with herpes simplex virus, respiratory syncytial virus and adenovirus were subjected to several cryogenic regimens to determine whether these procedures were detrimental to the performance and results obtained with an indirect immunofluorescence assay (IFA). Infected cells were harvested and divided into equal aliquots prior to freezing at different temperatures (-20, -70 and -165 degrees C). Cells fixed onto glass slides immediately after harvesting were used as controls. For the IFA, cells were thawed and fixed onto glass slides immediately prior to reaction with an appropriate virus-specific antibody and, subsequently, an FITC-conjugated anti-species antibody. The virus-infected cells retained their structural integrity following the experimental cryogenic regimens and were useable in the IFA. The quality of the image and the intensity of the fluorescent signal were enhanced by the incorporation of dimethyl sulfoxide into the freezing medium; cells frozen at -165 degrees C with DMSO gave the most satisfactory IFA results, although cells frozen at either -20 or -70 degrees C with DMSO were also useable. The use of cells frozen at the experimental temperatures in either phosphate buffered saline or tissue culture medium resulted in IFA substrate slides of diminished quality, often with a blurred image and distorted cellular morphology. We conclude that freezing of virus-infected cells prior to preparation for IFA is not detrimental to the performance of this procedure and the interpretation of immunological reactivity.     

Factors affecting viability and growth in HeLa 229 cells of Chlamydia sp. strain TWAR.

Two prototype isolates (TW-183 and AR-39) of Chlamydia sp. strain TWAR were used to study factors affecting growth of this organism in HeLa 229 cells. The results showed that an incubation temperature of 35 degrees C was better than one of 37 degrees C for growth. The burst size after 3 days of incubation at 35 degrees C was found to be small (13 to 52), which partially explains the difficulty of serial passage in cell culture. Application of a higher centrifugal force (1,700 X g versus 900 X g) at the time of inoculation enhanced growth 2.2 to 3.6 times. Infectivity was enhanced by treatment of cells with DEAE-dextran (2.4 times) or poly-L-lysine (1.6 times), but not with Polybrene or polyethylene glycol. The viability of the TWAR organism in chlamydia transport medium SPG was also studied. It was shown that the organism was rapidly inactivated at room temperature (22 degrees C); only 1% remained viable after storage for 24 h. The viability was preserved at 4 degrees C, and 70% remained viable after storage for 24 h. Freezing at -75 degrees C inactivated 23% of the organisms when the organisms were frozen within 4 h after harvesting and stored at 4 degrees C before freezing.     

Freezing Adipose Tissue Grafts May Damage Their Ability to Integrate into the Host

In this study, the effect of freezing on the morphology, viability, and VEGF synthesis of human adipose tissue grafts is examined. Currently, storage of adipose grafts involves freezing in simple saline solutions. However, the effect of freezing on the morphology and function of adipose tissue remains unclear. As a result, this study attempts to determine whether freezing adipose grafts should be considered prior to soft-tissue augmentation. In this study, the freezing of adipose grafts in saline for only 24 hr resulted in morphological changes in vivo and affected their ability to synthesize VEGF. The use of a simple cryopreservation medium containing sucrose appeared to maintain VEGF synthetic levels by the grafts and improved both their morphology and retention in vivo. However, the benefits of this cryopreservation medium were directly linked to storage time as long-term storage did not result in any noticeable benefit to graft retention. Finally, as an alternative to freezing, adipose grafts were combined with human adipose-derived stem cells (ASCs) to determine if their presence could enhance in vivo graft structure. The presence of ASCs did appear to improve graft structure in vivo over the short term and was also capable of improving tissue morphology when combined with grafts frozen in PBS. In conclusion, the successful use of adipose grafts may require a closer examination of the graft's storage conditions and time. Specifically, it now appears that the practice of freezing in saline may not be advisable if graft viability, activity, and structure are to be maintained in vivo.     

Cryopreservation of artificial cartilage: viability and functional examination after thawing

In biomedical research and in reconstructive surgery, preservation of intact tissue has been an unsolved problem. In this study, we investigated the viability of cryopreserved artificial cartilage and its synthetic activity of cartilage-specific matrix proteins after thawing for in vitro use. A polymer fleece cylinder (diameter = 3 mm; height = 3 mm) was loaded with a suspension of bovine chondrocytes (25 x 10(6)/ml) and encapsulated with fibrin glue. After a culture period of 1 week, the artificial cartilage units were frozen in a cryoprotection solution containing 10% basal medium (RPMI 1640), 10% DMSO and 80% FCS. The freezing procedure consisted of three steps: a 30-min period at +4 degrees C followed by a 24-hour storage at -80 degrees C. After that, the tissue units were transferred into liquid nitrogen (-196 degrees C) for final storage. Using histochemical staining techniques of cryogenic slices, we investigated the ability of cryopreserved artificial cartilage to produce its specific matrix after thawing. A modified MTT assay was used to determine the viability of frozen tissue units in comparison with unpreserved samples at different moments after thawing. Depending on the chondrocytes used for the formation of artificial cartilage, the viability of cryopreserved tissue varied between 65 and 85%. Both the intensity of alcian blue staining for proteoglycans and the azan staining for collagens increased proportionally with incubation time after thawing. These findings indicate that cryopreservation of small artificial cartilage units is possible with a minor loss of cell viability. Secondly, its synthetic activity of cartilage-specific matrix did not decline after the freezing process.     

Maximizing the retention of antigen specific lymphocyte function after cryopreservation

The ability to cryopreserve lymphocytes in peripheral blood mononuclear cells (PBMC) to retain their function after thawing is critical to the analysis of cancer immunotherapy studies. We evaluated a variety of cryopreservation strategies with the aim of developing an optimized protocol for freezing and thawing PBMC to retain viability and function. We determined several factors which do not affect cell viability after cryopreservation such as shipping frozen samples on dry ice, the length of time and speed at which samples are washed and centrifuged after thawing, and the number of cells frozen per container. Different media additives, however, did impact the viability of the cells after thawing. There was a significant reduction in the viability of the cells after freezing when using human AB serum compared to all other additives tested (p<0.000). A second critical parameter was the temperature of the media used to wash the cells after removal from the cryotubes. When the media was cooled to 4 degrees C prior to washing, the mean viability was 69.7+/-12.5%, at 25 degrees C 92.55+/-3.1%, and at 37 degrees C 95.11+/-2.5%. Finally, we used an optimized cryopreservation protocol with different media additives to determine if functional T cell responses to tetanus toxoid could be preserved. There was a statistically significant correlation between the tetanus specific stimulation index (S.I.) of the non-cryopreserved PBMC and SI obtained from cells frozen with media containing human serum albumin as compared to other additives such as dextran or fetal bovine serum.     

Assessment of viability and osteogenic ability of human mesenchymal stem cells after being stored in suspension for clinical transplantation

Human mesenchymal stem cells (MSCs) were suspended in phosphate-buffered saline (PBS) and stored up to 24 h at 4 degrees C, 24 degrees C, and 37 degrees C. More than 80% viability was maintained at any temperature for at least 1 h, then gradually decreased over time. After 24 h, the viabilities at 4 degrees C, 24 degrees C, and 37 degrees C were about 81%, 70%, and 62%, respectively. The MSCs suspended/stored in PBS at 4 degrees C for 24 h also exhibited in vitro osteogenic differentiation capability as evidenced by mineralized matrix formation as well as high alkaline phosphatase activity when cultured in an osteogenic medium. Furthermore, in vivo implantation experiments using the MSCs also demonstrated new bone formation. Because MSCs are known to possess multipotential stem cell characteristics, these data indicate that human MSCs stored in PBS at 4 degrees C could be delivered to distant medical facilities for the purpose of hard tissue and other types of tissue regeneration therapy.     

The rapid cooling contracture of toad cardiac muscles

The effect of rapid cooling on the mechanical activities of the cardiac muscles of the Japanese toad was examined. 1. The mechanical responses produced by rapid cooling (23 leads to 0 degrees C) in both auricular and ventricular muscle preparations were composed of an immediate effect (phasic contraction) and a delayed effect (tonic contraction). The immediate effect was blocked by TTX, but the delayed effect was not affected by TTX and Mn. 2. The tonic contraction of the delayed effect produced by rapid cooling was potentiated in Na-deficient Ringer solution in which an equivalent amount of NaC1 was substituted for Tris-C1. The relationship between the relative tension of the delayed effect produced by rapid cooling and the ratio of [Ca2+]/[Na+]2 showed an S-shaped curve. 3. Generation of tension development of the delayed effect produced by rapid cooling was obviously dependent upon the extracellular Ca concentrations. There waa an S-shaped relationship between the relative tension of the delayed effect and pCa in the extracellular medium. 4. In the test solution containing Ca less than pCa8.0, no tension development of the delayed effect was observed. However, the rapid cooling contracture was strongly elicited by application of caffeine (caffeine-RCC). 5. The caffeine-RCC was also observed in the presence of 100 mM KC1. 6. From the results of the present study, it was considered that, even though the Ca influx was abolished, the contraction of the cardiac muscle could be produced by another mechanism, probably due to increase of myoplasmic Ca released by raid cooling from the cell membrane itself and the sarcoplasmic reticulum.     

Effect of freezing on the viability of Mycobacterium paratuberculosis in bovine feces.

Three bovine fecal specimens were cultured for Mycobacterium paratuberculosis before freezing and after frozen storage at -70 degrees C for 3 and 15 weeks. The losses in viability from 0 to 3 weeks of storage were significant (P = 0.01) for all three samples. The losses in viability between 3 and 15 weeks of storage were not significant (P greater than 0.05) for two specimens in which the M. paratuberculosis occurred naturally, but the loss was significant (P = 0.01) in a simulated specimen (M. paratuberculosis culture added to feces from a healthy cow).     

An efficient method for the cryopreservation of fetal human liver hematopoeitic progenitor cells

The use of human hematopoietic progenitor cells (HPC) for transplantation requires efficient recovery methods and cryopreservation procedures. The purpose of this study was to determine cryopreservation techniques for fetal human liver (FHL) CD34(+) cells. We assessed FHL HPC recovery efficiency after freezing and thawing by viability testing, fluorescence-activated cell sorting analysis, and colony-forming ability under different conditions. We also determined optimal cell freezing concentrations and the effect of rate-controlled freezing on cell recovery. Lastly, cell recovery after varying freezing time periods was examined. Our results indicated that optimal cell recovery occurs when: A) cryopreservation medium consists of either 5% dimethylsulphoxide (DMSO) or 10% DMSO in combination with either 20% fetal bovine serum (FBS) or 70% FBS and when Iscove's modified Dulbecco's medium consists of not more than 10% DMSO; B) a rate-controlled freezing device container is used; C) CD34(+) cells are frozen at a concentration of 1 x 10(6)/ml, and D) a thawing temperature of 37 degrees C is used. These observations indicate that cryopreservation of FHL HPC is possible for up to 18 months in optimal conditions without losing hematopoietic activity.     

Studies on the procurement of blood coagulation factor VIII: effects of plasma freezing rate and storage conditions on cryoprecipitate quality.

Plasma was frozen and stored in different ways before processing to cryoprecipitate by a standard thawing technique. Freezing rate was found to be important with slow freezing having a deleterious effect on cryoprecipitate quality. Storage of frozen plasma at constant temperatures for periods up to six months had no effect on the quality of cryoprecipitate, with no difference being found for plasma stored at -20 degrees C or -40 degrees C. Subjecting frozen plasma to deliberate temperature fluctuations resulted in a considerable increase in the amount of fibrinogen recovered in cryoprecipitate, with the factor VIII yield being relatively unaffected.     

Some observations on cooling in laboratory autoclaves.

Cooling times for various autoclave loads were recorded using thermocouples. Loads which may be encountered during normal laboratory working require between 17 and 174 minutes to cool to 100 degrees, C and up to 260 minutes to cool to 95 degrees C. Such long cooling times had an adverse effect on nutritive properties of bacteriological culture medium. Observations are made on some factors which influenced the cooling times, and the need for artificially assisted cooling is stressed. Recommendations are given for the safe operation of laboratory autoclaves.     

Improvement in rabbit corneal cell suspension viability after freezing with Gingko Biloba extract

We investigated whether the addition of Gingko Biloba extract (EGb 761) to rabbit corneal epithelial medium before cell freezing improved cell viability after freezing then thawing. After removal of corneas, they were treated with enzymes and the corneal epithelium was prepared as a single cell suspension in freezing media with or without EGb 761. After freezing for two weeks then thawing, a higher cell viability was found in the cornea cell suspensions which had been frozen pretreated with EGb 761 in the media. The improvement with corneal cell viability with EGb 761 pretreatment is postulated to be based on the antioxidant capacity of the plant extract.     

Tailoring the pore architecture in 3-D alginate scaffolds by controlling the freezing regime during fabrication

The pore architecture in 3-D polymeric scaffoldings plays a critical role in tissue engineering as it provides the framework for the seeded cells to organize into a functioning tissue. In the present paper, we investigate the effect of freezing regime on the pore microstructure in 3-D alginate scaffolds, fabricated by the freeze-dry method. The scaffolds have shown isotropic pore structure, when the calcium crosslinked alginate solutions were slowly frozen at -20 degrees C, in a nearly homogenous cold atmosphere; the pores were spherical and interconnected. In contrast, when the cooling process was performed in liquid nitrogen or oil bath, where a temperature gradient was formed along the freezing solution, two main regions of pore structure were noted; at the interface with the cooling medium, small spherical pores were seen and above them a region with elongated pores. The different pore shape affected the compressibility of the scaffolds, while it had no effect on albumin diffusion. Rat hepatocytes seeded within the scaffolds were arranged according to the their pore shape. In scaffolds with elongated pores, the cells were lining along the pores, thus forming lines of interacting cells. In the scaffolds with the isotropic spherical pores, the hepatocytes clustered into spheroid-like aggregates. Thus, it appears that pore shape can modulate hepatocyte morphogenesis.     

小鼠精原干细胞冷冻保存

目的 探索小鼠精原干细胞(SSCs)冷冻保存方法 以及解冻后体外快速增殖的条件. 方法 实验以6d龄雄性昆明小白鼠为材料,两步酶消化法分离小鼠睾丸生殖细胞,Percoll非连续密度梯度离心法富集小鼠精原干细胞,随后加入不同的冷冻液以及采用不同的降温速率冷冻小鼠精原干细胞.以MEMα为基本培养基,加入10%胎牛血清和100μg/L的胶质细胞源性神经营养因子, WST-8比色法分析培养SSCs 复苏后的增殖率,运用碱性磷酸酶细胞化学染色和RT-PCR技术,对培养的SSCs进行鉴定. 结果 冷冻液中添加10%二甲基亚砜、10%胎牛血清、0.07mol/L蔗糖时,以1℃/min程控降温方式冷冻小鼠SSCs,细胞解冻后活率最高,达84%以上;采用非程控降温方式冻存SSCs,尽管细胞解冻后活率相对于程控降温方式略低,但具有方法 简单、易于操作、无需昂贵仪器的优点,复苏后SSCs贴壁时间为8～12h,24h可见细胞分裂,48h细胞出现迅速增殖,96h可见较多含20～25细胞的细胞团,此时精原干细胞增殖近5倍. 结论 本实验所用培养条件,可以使经长期冷冻的SSCs短期快速增殖.     

Cryopreservation of stem cells using trehalose: evaluation of the method using a human hematopoietic cell line

While stem cell cryopreservation methods have been optimized using dimethylsulfoxide (DMSO), the established techniques are not optimal when applied to unfertilized human embryonic cells. In addition, important questions remain regarding the toxicity and characteristics of DMSO for treatment of stem cells for clinical use. The objective of this study was to establish an optimal method for cryopreservation of stem cells using low concentrations (0.2 M) of trehalose, a nontoxic disaccharide of glucose, which possesses excellent protective characteristics, in place of current methods utilizing high concentrations (1-2 M) of DMSO. A human hematopoietic cell line was used in this investigation as a surrogate for human stem cells. Trehalose was loaded into cells using a genetically engineered mutant of the pore-forming protein alpha-hemolysin from Staphylococcus aureus. This method results in a nonselective pore equipped with a metal-actuated switch that is sensitive to extracellular zinc concentrations, thus permitting controlled loading of trehalose. Preliminary experiments characterized the effects of poration on TF-1 cells and established optimal conditions for trehalose loading and cell survival. TF-1 cells were frozen at 1 degrees C/min to -80 degrees C with and without intra- and extracellular trehalose. Following storage at -80 degrees C for 1 week, cells were thawed and evaluated for viability, differentiation capacity, and clonogenic activity in comparison to cells frozen with DMSO. Predictably, cells frozen without any protective agent did not survive freezing. Colony-forming units (CFU) generated from cells frozen with intra- and extracellular trehalose, however, were comparable in size, morphology, and number to those generated by cells frozen in DMSO. There was no observable alteration in phenotypic markers of differentiation in either trehalose- or DMSO-treated cells. These data demonstrate that low concentrations of trehalose can protect hematopoietic progenitors from freezing injury and support the concept that trehalose may be useful for freezing embryonic stem cells and other primitive stem cells for therapeutic and investigational use.     

"Wet-state" mechanical properties of three-dimensional polyester porous scaffolds

Porous poly(D,L-lactic-co-glycolic acid) (PLGA) scaffolds under a simulated physiological environment were investigated to estimate their "true" mechanical properties, with emphasis on the effect of "wet-state" on the compressive behaviors. The effect of the history of ethanol sterilization was also investigated. The studies were focused upon the "wet-state" mechanical properties of polyester porous scaffolds, because the potential implants must be used under a wet environment. The measurements of three-dimensional porous scaffolds composed of amorphous PLGA with five polymer formulations including poly(D,L-lactic acid) (PDLLA) demonstrated that the mechanical properties of PLGA scaffolds significantly decreased in phosphate buffer saline solution (PBS) at 37 degrees C and/or with an ethanol sterilization history, even though PLGA is a hydrophobic material. The decrease extent depends on the copolymer composition: when the porosity is about 90%, a PDLLA scaffold remained about 75-80% of initial mechanical properties in the dry state at 25 degrees C, whereas PLGA 85:15, 75:25, and 65:35 scaffolds remained only about 10% or less, and the PLGA 50:50 scaffolds examined were not sufficiently strong for mechanical tests. If scaffolds were prewetted with ethanol ahead of prewetting with PBS, the mechanical properties further decreased compared with those merely prewetted with PBS. These phenomena were elucidated experimentally from plasticization of PLGA with water or ethanol, and the consequent reduction of glass transition temperature. The results might be helpful for designing polyester porous scaffolds for tissue engineering or in situ tissue induction applications.