Cryopreservation of the common carotid artery of the rabbit: Optimization of dimethyl sulfoxide concentration and cooling rate.

This paper describes the continuation of studies that demonstrated the suitability of CP-Tes solution as a medium for the introduction and removal of dimethyl sulfoxide in rabbit common carotid arteries and established the kinetics of cryoprotectant permeation in that tissue. In this paper we report the tolerance of rabbit common carotid artery to dimethyl sulfoxide, in concentrations up to 30% (w/w), using a technique of exposure that was designed to control osmotic stress. The maximum concentration achieved without damage was 15% (w/w). Vessels were then equilibrated with 15% dimethyl sulfoxide and cooled to -80 °C at 0.22, 0.69, 2.15, or 9.63 °C/min: they were then transferred to the gas phase of a liquid nitrogen refrigerator {temperature below -160 °C) for storage. Thawing was carried out in a 37 °C water bath. The optimum rate of cooling for these conditions was found to be 0.69 °C/min. The maximal recovery of contractile force in response to 10-6 M norepinephrine was 30-40%; relaxation to acetylcholine (an endothelium-mediated function) was 80% of control, and an estimated 71% of endothelial cells survived with minimal ultrastructural change.

EVALUATION OF DMSO TRANSPORT IN HUMAN ARTICULAR CARTILAGE: VEHICLE SOLUTIONS AND EFFECTS ON CELL FUNCTION.

Osteochondral allografting techniques are limited by the availability of suitable donor tissue; there is an urgent need for effective cryopreservation. A fundamental requirement is the need to establish initial conditions of exposure to cryoprotectant that the chondrocytes will tolerate and that load the tissue with an adequate concentration of cryoprotectant. Three vehicle solutions to transport DMSO into the tissue were studied. Knee joints were obtained from deceased donors with appropriate consent. Whole condyles were treated with 20% w/w DMSO in each of three vehicle solutions and chondrocyte function and tissue CPA content measured. The results showed that exposure to 20% DMSO in each vehicle solution for 2 hours at 0 degrees C was tolerated without loss of GAG synthetic activity. It was observed that penetration of DMSO increased little after 1 hour of CPA exposure at 0 degrees C but the final tissue concentration of CPA was markedly lower than that in the medium.

A liquidus tracking approach to the cryopreservation of human cartilage allografts.

In the "liquidus tracking" (LT) approach to cryopreservation both the temperature and the concentration of cryoprotectant (CPA) are controlled such that solution composition "tracks" the liquidus (melting point) line for that system. Ice crystal formation is prevented but the tissue is not exposed to CPA concentrations exceeding those experienced by cells during conventional cryopreservation. This approach is particularly appropriate for articular cartilage because chondrocytes in situ are exquisitely susceptible to damage by the crystallisation of ice. This project aimed to develop a suitable process for tissue to be used in the surgical repair of damaged human knee joints. A high proportion of the chondrocytes should be alive. Human articular cartilage was obtained from deceased donors and dimethyl sulphoxide (DMSO) was used as the CPA, cooling was at 0.14°C/min and warming at 0.42°C/min. The vehicle solution was CPTes2. A program of increasing DMSO concentration was developed for cooling and this gave satisfactory tissue concentrations but reduction of DMSO concentration during warming was inadequate, resulting in higher tissue concentrations than required. Biomechanical testing indicated a compressive modulus of 9.5±1.3 MPa in LT-processed cartilage, with control values of 11.6±0.8 MPa (p>0.05, Student's t-test). Measurement of GAG synthesis sometimes approached 65% or 85% of control, but the variability of replicate data prevented firm conclusions. Ideally allograft tissue should score 1A or above on the Noyes scale and the donor age should be less than 46 years but the cartilage used in this study did not meet these standards.

A standardised protocol for the validation of banking methodologies for arterial allografts.

The objective of this study was to design and test a protocol for the validation of banking methodologies for arterial allografts. A series of in vitro biomechanical and biological assessments were derived, and applied to paired fresh and banked femoral arteries. The ultimate tensile stress and strain, suture pullout stress and strain, expansion/rupture under hydrostatic pressure, histological structure and biocompatibility properties of disinfected and cryopreserved femoral arteries were compared to those of fresh controls. No significant differences were detected in any of the test criteria. This validation protocol provides an effective means of testing and validating banking protocols for arterial allografts.

Application of a high-level peracetic acid disinfection protocol to re-process antibiotic disinfected skin allografts.

Skin allografts, derived from cadaveric donors, are widely used for the treatment of burns and ulcers. Prior to use in clinical situations, these allografts are disinfected using a cocktail of antibiotics and then cryopreserved. Unfortunately, this antibiotic disinfection procedure fails to decontaminate a significant proportion and these contaminated grafts can not be used clinically. We have investigated whether it is possible to apply a second, more potent disinfection procedure to these contaminated grafts and effectively to re-process them for clinical use. Cadaveric skin grafts, treated with antibiotics and cryopreserved, were thawed and a peracetic acid (PAA) disinfection protocol applied. The grafts were then preserved in a high concentration of glycerol or propylene glycol, and properties thought to be essential for successful clinical performance assessed. The cytotoxicity of the grafts was assessed using both extract and contact assays; damage to the skin collagen was assessed using a collagenase susceptibility assay and the capacity of the grafts to elicit an inflammatory response in vitro was assessed by quantifying the production of the pro-inflammatory cytokine TNF-alpha by human peripheral blood mononuclear phagocytes. PAA disinfection, in conjunction with either glycerol or propylene glycol preservation, did not render the grafts cytotoxic, pro-inflammatory, or increase their susceptibility to collagenase digestion. The rates of penetration of glycerol and propylene glycol into the re-processed skin were comparable to those of fresh skin. This study has demonstrated that PAA disinfection combined with immersion in high concentrations of either glycerol or propylene glycol was an effective method for re-processing contaminated skin allografts, and may justify their clinical use.

Permeability characteristics and osmotic sensitivity of rhesus monkey (Macaca mulatta) oocytes.

BACKGROUND: Permeability characteristics and sensitivity to osmotic shock are principal parameters that are important to derive procedures for the successful cryopreservation of mammalian oocytes. METHODS AND RESULTS: The osmotically inactive volume of rhesus monkey oocytes was determined by measuring their volumes in the presence of hypertonic solutions of sucrose from 0.2 to 1.5 mol/l, compared with their volume in isotonic TALP-HEPES solution. Boyle-van't Hoff plots at infinite osmolality indicated that the non-osmotic volumes of immature and mature oocytes were 20 and 17% respectively. Osmotic responses of oocytes exposed to 1.0 mol/l solutions of glycerol, dimethylsulphoxide (DMSO) and ethylene glycol (EG) were determined. Rhesus monkey oocytes appeared to be less permeable to glycerol than to DMSO or to EG. Sensitivity of oocytes to osmotic shock was determined by exposing them to various solutions of EG (0.1 to 5.0 mol/l) and then abruptly diluting them into isotonic medium. Morphological survival, as measured by membrane integrity, of oocytes diluted out of EG depended significantly on the concentration of EG (P < 0.01). CONCLUSION: Determination of permeability characteristics and sensitivity to osmotic shock of rhesus oocytes will aid in the derivation of procedures for their cryopreservation.

The history and principles of cryopreservation.

The ability of glycerol to protect cells from freezing injury was discovered accidentally. The subsequent development of cryopreservation techniques has had a huge impact in many fields, most notably in reproductive medicine. Freezing injury has been shown to have two components, direct damage from the ice crystals and secondary damage caused by the increase in concentration of solutes as progressively more ice is formed. Intracellular freezing is generally lethal but can be avoided by sufficiently slow cooling, and under usual conditions solute damage dominates. However, extracellular ice plays a major role in tissues. Cryoprotectants act primarily by reducing the amount of ice that is formed at any given subzero temperature. If sufficient cryoprotectant could be introduced, freezing would be avoided altogether and a glassy or vitreous state could be produced, but osmotic and toxic damage caused by the high concentrations of cryoprotectant that are required then become critical problems. The transport of cryoprotectants into and out of cells and tissues is sufficiently well understood to make optimization by calculation a practical possibility but direct experiment remains crucial to the development of other aspects of the cryopreservation process.

Processing of ovine cardiac valve allografts: 1. Effects of preservation method on structure and mechanical properties.

It is essential to have some method of preservation of allograft valves during the time between procurement and implantation. Cryopreservation is the most commonly-used storage method today but it has the major disadvantage of high cost, and because its aim is to preserve living cells only relatively gentle antimicrobial treatments are used. This study addresses two interrelated questions: Is it necessary to maintain living donor cells in the tissue graft? Can more effective measures be used to reduce the risk of transmission of diseases, especially viral diseases, via human tissue grafts. In this paper, we report an investigation of four preservation methods that could be combined with more effective disinfection: cryopreservation with dimethyl sulphoxide, storage at approximately 4 degrees C in a high concentration of glycerol as used for the preservation of skin, snap-freezing by immersion in liquid nitrogen and vitrification. Snap freezing was mechanically damaging and vitrification proved to be impracticable but two methods, cryopreservation and storage in 85% glycerol, were judged worthy of further study. Cryopreservation was shown to maintain cellular viability and excellent microscopic structure with unchanged mechanical properties. The glycerol-preserved valves did not contain any living cells but the connective tissue matrix and mechanical properties were well preserved. The importance of living cells in allograft valves is uncertain. If living cells are unimportant then either method could be combined with more effective disinfection methods: in that case the simplicity and economy of the glycerol method would be advantageous. These questions are addressed in the two later papers in this series.

Processing of cardiac valve allografts: 2. Effects of antimicrobial treatment on sterility, structure and mechanical properties.

This is the second in a series of papers that report experiments to investigate the properties required for effective tissue valve implants. This paper is concerned with investigations into alternative antimicrobial treatments and the effect these treatments produce on the structural and biomechanical properties of ovine aortic valves. Six treatments were studied: heat, peracetic acid (at two concentrations), chlorine dioxide, a surfactant cleaning agent and a solvent/detergent treatment. Samples of myocardial tissue were exposed to a mixed bacterial culture or one of three virus cultures and then decontaminated. Two of the six treatments (0.35% peracetic acid and heat) were effective in removing both bacterial and viral contamination, reducing levels of contamination by 2.5 to 3 logs, whilst a third (chlorine dioxide) was effective against viruses ( approximately 3 log reduction). Valves subjected to these treatments were examined by microscopy and measurements of mechanical properties were made. All three treatments seriously damaged endothelial cells and leaflet fibroblasts. Heat treatment also damaged connective tissue components (collagen and elastin) but these changes were not seen after chemical treatment. Mechanical testing confirmed severe damage following heat treatment but chemical treatment showed only minor effects on the elasticity of the leaflets and none on extensibility. These minor effects could be mitigated by exposure to a lower dose of peracetic acid and this treatment could be safely combined with cryopreservation or storage in 85% glycerol. Peracetic acid was the preferred disinfection method for use in the subsequent in vivo studies in sheep.

Processing of ovine cardiac valve allografts: 3. Implantation following antimicrobial treatment and preservation.

It is known that a satisfactory clinical outcome can follow the implantation of cardiac valve allografts in spite of the loss of living cells in the tissue. If viable cells are not required for long term graft function, then effective disinfection of the tissue might become possible. In an earlier paper in this series we reported that peracetic acid (PAA) is an effective antimicrobial agent for the treatment of valve allografts; it was lethal to the cells but at a concentration of 0.21% had little effect on the mechanical properties or extracellular morphology of the valve leaflets. It was also found that PAA-treatment could be combined with storage in 85% glycerol at 4 degrees C, or cryopreservation with 10% Me(2)SO, without substantial further impairment of microscopic structure or mechanical properties. In this paper we describe the implantation of processed ovine aortic valves in the descending thoracic aorta of sheep. The experimental groups included control untreated valves and valves that had been treated with antibiotics or PAA and either cryopreserved, or stored in 85% glycerol. The recipient sheep showed good clinical appearances until the experiment was terminated at six months. The explanted grafts were examined by standard morphological and mechanical testing methods. The PAA-treated valves were clearly recognisable as valves: the leaflets had fair to medium morphology in both the unpreserved and the cryopreserved groups. All leaflets had a superficial overgrowth of cells. Microsatellite analysis for allelic differences were performed on samples of donor and recipient tissues using three markers of tissue source. Only one valve, which had been treated with PAA, revealed allelic differences between donor and recipient. It is suggested that DNA-fragments may have remained after the destruction of donor cells and six months of implantation: the overgrowing cells were almost certainly of recipient origin. We conclude that our experiments, in which PAA-treatment was combined with preservation, are sufficiently encouraging to justify further studies to refine the technique, but in our opinion they are not sufficient to justify a clinical trial at this time.

Differences in the requirements for cryopreservation of porcine aortic smooth muscle and endothelial cells.

One of the basic requirements for the production of tissue-engineered constructs is an effective means of storing both the constructs and the cells that will be used to make them. This paper reports on the cryopreservation of porcine aortic smooth muscle and endothelial cells intended for the production of model vascular constructs. We first determined the cell volume, nonosmotic volume, and the permeability parameters for water and the cryoprotectant dimethyl sulfoxide (Me(2)SO) in these cells at 2-4 degrees and 22 degrees C. The following results were obtained: Table unavailable in HTML format. Using a cell culture assay, both cell types were shown to tolerate threefold changes in cell volume, in either direction, without significant injury. Although these data suggested that single-step methods for the introduction and removal of 10% w/w Me(2)SO should be effective, an additional mannitol dilution step was adopted in order to reduce the time required for removal of the Me(2)SO. Following cooling at 0.3, 1, or 10 degrees C/min and storage at less than -160 degrees C, the survival of porcine aortic smooth muscle cell suspensions, measured by a cell culture assay, was inversely related to cooling rate; at 0.3 degrees C/min, recovery was >80%. The survival rate for aortic endothelial cells was directly related to cooling rate over the range tested and was >80% at 10 degrees C/min.

The current status of tissue cryopreservation.

Cryopreservation plays an important role in tissue banking and will assume even greater importance when tissue engineering becomes an everyday reality. For some tissue grafts, living cells are unnecessary and adequate preservation methods are usually available. For other tissues living and functioning cells are needed and preservation methods are much less advanced. The basic requirements for cell recovery can usually be defined if a few basic biophysical properties of the cell are known and some standard measurements of the effect of cryobiological variables are carried out. The problems in tissue cryopreservation are not usually due to difficulties in preserving the living cells per se, but arise from the properties of the integrated cell/matrix systems upon which tissue function almost always depends. Some examples of such difficulties are described. It is concluded that the formation of ice, through both direct and indirect effects, is probably fundamental to these difficulties, and this is why vitrification seems to be the most likely way forward. However, two major problems still to be overcome are cryoprotectant toxicity and recrystallization during rewarming. Less obvious, and certainly less well understood is chilling injury - damage caused by reduction in temperature per se; this may yet turn out to be of fundamental importance.

The banking of arterial allografts in the United kingdom. A technical and clinical review.

The standard graft materials for reconstructive vascular surgery are autologous vessels and synthetic prostheses. However, the clinical results are unsatisfactory when the diameter of the required graft is less than 6 mm and attention has therefore been directed to the possibility of using vascular allografts when autologous conduits are not available. In this review, we consider the evidence that cryopreserved allogeneic vessels might meet this need. Other factors being equal, the literature suggests that arteries will give superior performance to veins when grafted into the arterial circulation. Conventional cryopreservation techniques have been developed and these can provide structurally intact and functional small elastic arteries in animal experiments. We conclude that cryopreservation per se produces only modest injury which may well be recoverable following grafting. However, both experimental and clinical studies suggest that immunological injury is a greater problem. Nevertheless, the general indication is that appropriate risk-benefit analysis in individual cases will justify the clinical use of cryopreserved allograft vessels. Immunosuppression with careful immunological monitoring may be justified in the more desperate clinical situations. Finally we conclude that the existing tissue bank network in the United Kingdom is well placed to provide surgeons with such tissue.

Rapid electromagnetic warming of cells and tissues.

We describe a system for thawing frozen cell suspensions and tissues by electromagnetic absorption. A 25-ml sample is heated in a cylindrical resonant cavity, which is excited in three modes all close to 434 MHz. Maximum warming rates are over 10 degrees C/s (600 C/min), and a frozen sample may be brought from -65 degrees C to room temperature in < 30 s, with final spatial differences of < 20 degrees C. Samples may be frozen externally, or cooled within the cavity at typically 1 degree C/min. We have also used the resonant cavity to measure the permittivity and conductivity of the sample at temperatures from -83 degrees C to +8 degrees C. By measuring the heat capacity of the sample, we have calculated the power deposited in it as a function of its temperature. The system is currently being used to investigate the effect of warming rate on cell survival.

Cryopreservation studies with porcine corneas.

PURPOSE: A new technique for the cryopreservation of rabbit corneas in 20% w/w dimethylsulfoxide, which has been shown to preserve significant structural and functional integrity of the endothelium, was tested in porcine corneas. METHODS: The characteristics of uptake of dimethylsulfoxide into porcine corneas were measured using proton ( 1 H) nuclear magnetic resonance (NMR) spectroscopy. The effect on structural integrity of exposure to 20% w/w dimethylsulfoxide without freezing was first assessed using vital staining (acridine orange and propidium iodide), and optimum temperature conditions for addition and removal of the cryoprotectant were derived. The effects on structural integrity of cryopreservation in 15% and 20% w/w dimethylsulfoxide, and of reducing the degree of cell swelling during cryoprotectant removal following cryopreservation, were then evaluated. RESULTS: The characteristics of uptake of dimethylsulfoxide from a 10% w/w solution fitted a single exponential, resulting in a maximum tissue concentration of 14.6% when the addition occurred on ice, and 18.5% when the addition took place at room temperature. The toxic effects of dimethylsulfoxide in porcine corneas were highly temperature dependent and only evident after removal of the cryoprotectant. Unlike rabbit corneas, cryopreservation of porcine corneas in 15% and 20% w/w dimethylsulfoxide induced substantial endothelial injury which was not improved by reducing the degree of cell swelling that occurred during removal of the cryoprotectant. CONCLUSIONS: Porcine corneas were substantially more susceptible to the toxic effects of dimethyl sulfoxide, and to cryopreservation injury, than rabbit corneas. These results underline the importance of species variation in animal studies aimed at the cryopreservation of human tissue for transplantation.

Osmotically inactive volume, hydraulic conductivity, and permeability to dimethyl sulphoxide of human mature oocytes.

Controlled ovarian stimulation during an in vitro fertilization cycle usually produces large numbers of oocytes and, consequently, it is likely that more embryos will be generated than can be transferred in a given cycle. It is desirable to freeze-bank surplus oocytes before insemination to avoid the ethical and legal complications of disposing of or storing embryos. Although many attempts have been made to cryopreserve human oocytes, to date, post-thaw survival has been poor, and viable pregnancies after in vitro fertilization have been rare. A possible explanation for the lack of success is that the freezing methods have been adapted from animal studies but have not been optimized for the human oocyte. In this study, video microscopy was used to determine the volumetric responses of mature human oocytes to changes in osmolarity during preparation for freezing. A Boyle van't Hoff plot of data collected in static experiments with fresh human oocytes gave a value of 0.19 +/- 0.01 (mean +/- SEM) for the osmotically inactive volume. Dynamic measurements during exposure to dimethyl sulphoxide at room temperature (22 degrees C) were analysed by a two-parameter transport model and produced values of 1.30 x 10(-6) cm atm-1 s-1 for the hydraulic conductivity of the plasma membrane and 3.15 x 10(-5) cm s-1 for dimethyl sulphoxide permeability (chi-squared = 0.43, df = 20) of fresh human oocytes. Oocytes that had failed to fertilize had a slightly lower hydraulic conductivity and dimethyl sulphoxide permeability and, after exposure to 1.5 mol dimethyl sulphoxide l-1, these cells appeared to become permeable to normally impermeable solutes. These permeability properties have been used to design a protocol for the addition and removal of dimethyl sulphoxide to control the magnitude of volumetric changes.

Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation.

The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or permanent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an important determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4 degrees C and 37 degrees C. We have also studied the effect of adding different concentrations of the non penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4 degrees C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37 degrees C all four cryoprotectants penetrated at a faster rate, however concern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4 degrees C; this achieved a mean tissue concentration that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.

Sensitivity of kidney perfusion protocol design to physical and physiological parameters.

The introduction and removal of cryoprotective agents (CPA) to a kidney via vascular perfusion may induce changes in cell volume that are destructive to the tubular epithelial or capillary endothelial cells as well as causing significant increases in vascular resistance that compromise the perfusion process. A network thermodynamic model of the coupled osmotic, hydrodynamic and elastic properties of the kidney was applied to evaluate the sensitivity of these critical outputs to a set of physiological and perfusion variables. Simulation results suggest that in the design of perfusion protocols for CPAs such as glycerol, it may be advantageous to: (a) select a CPA with as high a cell membrane permeability as possible; (b) increase the concentration of mannitol in the perfusate to about 200 mos/kg, beyond which there is no discernible benefit; (c) when glycerol is the CPA, limit the rate of reduction in the perfusate during removal to 30 mM/min or less; (d) limit the perfusion pressure to 20-30 mm Hg, within the practical constraints of the perfusion system; (e) increase the concentration of impermeant in the perfusate to as high as 400 mos/kg, although it is recognized that this departure from plasma-like composition might impose other problems that are not considered in this model. Further, it was observed that the vascular membrane permeability plays a relatively minor role in controlling cellular osmotic injury and vascular perfusion resistance and is therefore not a critical parameter in the perfusion design process.

Cryopreservation of rabbit corneas in dimethyl sulfoxide.

PURPOSE: To minimize the injury to endothelial cells during cryopreservation of rabbit corneas with dimethyl sulfoxide. METHODS: Rabbit corneas were cryopreserved using 20% wt/wt dimethyl sulfoxide (Me2SO), added and removed in stages to maintain the osmotically induced excursions in cell volume to within +/-40% of their isotonic volume. The vehicle solution, cooling rate, and conditions of storage used were those already reported to be optimal for endothelial cell survival after exposure to low temperatures. Survival was assessed by confocal microscopy with vital staining and by the ability of the endothelium to control stromal hydration during 3 hours of normothermic perfusion. The effect of temperature of addition and removal of Me2SO (room temperature [RT] or 2 degrees C) on endothelial viability also was measured. RESULTS: After thawing, all the cryopreserved corneas appeared structurally intact when assessed by vital staining and could limit stromal swelling during subsequent normothermic perfusion. Analysis of the rate of stromal swelling during the first 1.5 hours of normothermic perfusion indicated a substantial benefit when the Me2SO was removed at RT. Adding and removing the Me2SO at RT, which allowed a briefer exposure to Me2SO before cooling, resulted in better structural integrity of the endothelial layer than when the addition of cryoprotectant took place on ice. CONCLUSIONS: These results demonstrate the importance of osmotic stresses in the generation of injury to corneal endothelium during cryopreservation and the possibility of eventual successful cryopreservation of this tissue.

Repopulation of freeze-dried porcine valves with human fibroblasts and endothelial cells.

BACKGROUND AND AIMS OF THE STUDY: There is a need for a replacement cardiac valve constructed from non-immunogenic materials but incorporating living, and preferably autologous, cells. The object of this study was to colonize freeze-dried porcine valve leaflets with human fibroblasts and vascular endothelial cells. METHODS: Porcine pulmonary valve leaflets were freeze-dried to produce a porous matrix having communicating cavities of appropriate dimensions for fibroblast repopulation. Cultured human fibroblasts and vascular endothelial cells that had been cryopreserved by standard methods were added to freeze-dried leaflets. Following culture at 37 degrees C, the leaflets were examined by confocal scanning microscopy and transmission electron microscopy. RESULTS: Mechanical perforation of the leaflet surface permitted colonization of the freeze-dried matrix by fibroblasts; under the conditions we studied, the cell density did not reach physiologic levels but those cells that were present were well attached and metabolically active. Gentle cotton abrasion of the surface of the freeze-dried leaflets provided a suitable substrate for endothelial cell attachment and confluence was achieved in 10 days. Leaflets were perforated, cultured with human fibroblasts for 10 days, then gently rubbed with a cotton bud and cultured for a further 10 days with human endothelial cells. The endothelial cells formed a confluent layer on the surface and viable fibroblasts were present within the substance of the leaflet. CONCLUSION: Although these results are preliminary, they demonstrate the basic feasibility of this approach to the production of xenogeneic valves that contain the patient's own cells.