Presaturation of supercritical CO.SUB.2 .with water for decellularization of matrices

Abstract

Decellularization methods for tissue are provided. The method can include: exposing a tissue to a water-saturated, supercritical CO.sub.2. The method can further comprise, prior to exposing the tissue to the water-saturated, supercritical CO.sub.2, saturating a stream of supercritical CO.sub.2. The tissue can be exposed to the water-saturated, supercritical CO.sub.2 at a treatment temperature of about 35° C. to about 40° C. (e.g., about 37° C.). In one embodiment, the water-saturated, supercritical CO.sub.2 is completely saturated with water at the treatment temperature. The tissue can be exposed to the water-saturated, supercritical CO.sub.2 at a constant flow rate, such as less than 3 mL/min (e.g., about 0.5 mL/min to about 2.5 mL/min).

Claims

1. A decellularization method for tissue, the method comprising: passing a stream of supercritical CO.sub.2 through water retained in a first chamber at a flow rate of less than 3 mL/min and thereby forming a stream of water-saturated, supercritical CO.sub.2 that is completely saturated with water; feeding the stream of water-saturated, supercritical CO.sub.2 that is completely saturated with water into a treatment chamber, the treatment chamber containing a tissue, such that the tissue is exposed to the water-saturated, supercritical CO.sub.2 that is completely saturated with water; wherein the exposure causes decellularization of the tissue.

2. The decellularization method as in claim 1, wherein the tissue is exposed to the water-saturated, supercritical CO.sub.2 that is completely saturated with water at a treatment temperature of about 35° C. to about 40° C.

3. The decellularization method as in claim 2, wherein the tissue is exposed to the water-saturated, supercritical CO.sub.2 that is completely saturated with water at a treatment temperature of about 37° C.

4. The decellularization method as in claim 1, wherein the tissue is exposed to the water-saturated, supercritical CO.sub.2 at a constant flow rate.

5. The decellularization method as in claim 4, wherein the flow rate is about 0.5 mL/min to about 2.5 mL/min.

6. The decellularization method as in claim 1, wherein the water-saturated, supercritical CO.sub.2 that is completely saturated with water has a water content of about 0.004 mole fraction to about 0.009 mole fraction.

7. The decellularization method as in claim 6, wherein the water-saturated, supercritical CO.sub.2 has a water content of about 0.005 mole fraction to about 0.0084 mole fraction.

8. The decellularization method as in claim 1, wherein the tissue is exposed to the water-saturated, supercritical CO.sub.2 that is completely saturated with water at a treatment pressure of 2000 psi.

9. The decellularization method as in claim 1, wherein the tissue is exposed to the water-saturated, supercritical CO.sub.2 that is completely saturated with water for a treatment time of 30 to 60 minutes per 0.25 to 0.1 gram of tissue.

10. The decellularization method as in claim 1, further comprising following the decellularization, depressurizing the treatment chamber.

11. The decellularization method as in claim 10, wherein the treatment chamber is depressurized at a depressurization rate of 50 psi/min.

12. The decellularization method as in claim 1, wherein following decellularization, the weight of the tissue is from 95.4% to 98.1% of the weight of the tissue prior to decellularization.

13. The decellularization method as in claim 1, wherein the tissue exhibits less than a one percent average weight loss upon the decellularization.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) A full and enabling disclosure of the present invention, including the best mode thereof to one skilled in the art, is set forth more particularly in the remainder of the specification, which includes reference to the accompanying figures, in which:

(2) FIG. 1 shows a schematic of presaturation apparatus according to one particular embodiment;

(3) FIG. 2 shows a schematic of a cold trap utilized according to the examples;

(4) FIG. 3 shows the presaturation test results of the Examples;

(5) FIG. 4 shows an exemplary hydrogel test apparatus utilized in the Examples;

(6) FIG. 5 shows the comparison of the dry scCO.sub.2 and presaturated scCO.sub.2 hydrogel results at 37° C. and 50° C. according to the Examples;

(7) FIG. 6 shows the Porcine Aorta Drying Curve, 0-24 hours, according to the Examples;

(8) FIG. 7 shows the results of Vacuum Drying of Porcine Aorta, 0-6 hours, according to the Examples;

(9) FIG. 8 shows the results of the tissue dehydration comparison of the dry scCO.sub.2 and presaturated scCO.sub.2 results at 37° C. of the Porcine Aorta according to the Examples; and

(10) FIG. 9 shows the vacuum drying curve of the Porcine Aorta, compared to CO.sub.2, according to the Examples.

DEFINITIONS

(11) Chemical elements are discussed in the present disclosure using their common chemical abbreviation, such as commonly found on a periodic table of elements. For example, hydrogen is represented by its common chemical abbreviation H; helium is represented by its common chemical abbreviation He; and so forth.

DETAILED DESCRIPTION

(12) Reference now will be made to the embodiments of the invention, one or more examples of which are set forth below. Each example is provided by way of an explanation of the invention, not as a limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as one embodiment can be used on another embodiment to yield still a further embodiment. Thus, it is intended that the present invention cover such modifications and variations as come within the scope of the appended claims and their equivalents. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention, which broader aspects are embodied exemplary constructions.

(13) Methods and apparatus are generally provided for an effective scCO.sub.2-based decellularization method that overcomes certain limitations discussed above. In particular, the presently disclosed methods provide a major step in developing an effective scCO.sub.2-based decellularization method by preventing the tissue dehydration that occurred in the scCO.sub.2 decellularization experiments of Sawada et al. Because of the high water content in mammalian tissues (e.g., greater than 80% in a porcine aorta) maintenance of the natural hydration state of the tissue is important to fabricating a suitable TE scaffold.

(14) In the presently disclosed methods, the extraction of water is inhibited from occurring at all. In particular, a simple presaturation method is generally provided by using water-saturated scCO.sub.2. The presaturated scCO.sub.2 is then contacted with the tissue, but water is not substantially extracted from the tissue. That is, the presaturated scCO.sub.2 cannot dissolve any additional water due to the amount of water already within the presaturated scCO.sub.2 flow stream, since the amount of water presaturated can be close to or at the saturation limit of the scCO.sub.2. In particular embodiments, the flow rate can be relatively low, such as less than about 3 mL/min (e.g., about 0.5 mL/min to about 2.5 mL/min).

(15) Supercritical fluids, like gases, have little or no surface tension, low viscosity, and high diffusivity, but they also have liquid-like densities. This combination of properties allows supercritical fluids to penetrate through surfaces easily without damaging them. Upon depressurization, the supercritical fluids outgases leave no residue in the tissue. In particular, supercritical CO2 is especially useful in biomedical applications. It is inexpensive, readily available, chemically inert, and nontoxic. It has mild critical conditions of 31.1° C. and 7.38 MPa, making it able to treat biological materials at body temperature (37° C.) without reaching excessive pressures. Supercritical CO2 has already been shown to have applications in pasteurization, extraction, and sterilization.

(16) The ability of supercritical CO.sub.2 to dissolve small molecules, such as water, is well known. In physical chemistry, saturation is the point at which a solution of a substance can dissolve no more of that substance and additional amounts of it will appear as a separate phase. This point of maximum concentration (i.e., the saturation point) depends on the temperature and pressure of the solution, as well as the chemical nature of the substances involved. With respect to scCO.sub.2, the saturation point of water at 50° C. and 2000 psi (which is about 138 bar or 103,430 torr), the equilibrium solubility of water in CO.sub.2 is about 0.0084 mole fraction. At 37° C., the equilibrium solubility of water in CO.sub.2 would be a little lower, probably around 0.005 mole fraction.

(17) As such, the presently disclosed decellularization methods prevent tissue dehydration and removes all nuclear material from the tissue using liquid carbon dioxide (lCO.sub.2) or supercritical carbon dioxide (scCO.sub.2), while maintaining the tissue's mechanical integrity during the treatment process. Supercritical CO.sub.2 has the unique potential for accomplishing both decellularization and sterilization, which means that one comprehensive protocol may be possible.

EXAMPLES

(18) Here, the construction of an apparatus that can presaturate scCO.sub.2 with water is described to determine the range of flow rates where equilibrium-level presaturation can be attained and to compare the amount of water extraction caused by presaturated and dry CO.sub.2.

(19) 1. Apparatus Development and Validation

(20) First, a presaturation apparatus was developed to determine whether the proposed system could be used to effectively saturate scCO.sub.2 with water. The objective of using the apparatus was to ensure that the scCO.sub.2 was really being humidified and saturated during the mixing process, and that the flow rate used in Sawada's experiments (1 mL/min) was slow enough for presaturation to be maintained in a dynamic flow system. Confirming these findings was critical before proceeding to subsequent tests.

(21) The presaturation apparatus is shown in FIG. 1. Liquid carbon dioxide (bone-dry grade with siphon tube, 99.8% purity, Airgas National Welders, Charlotte, N.C.) was compressed to 2000 psi in a chilled syringe pump (500 HP Series, ISCO Inc, Lincoln, Nebr.), and slowly bubbled into a 25 mL stainless steel view cell (Waters Corp, Milford, Mass.) containing 10 mL of deionized (DI) water. The view cell was agitated using a stirrer plate and magnetic stirring bar, all of which were enclosed in an environmental chamber (LU-113 model, ESPEC Corp, Osaka, Japan), which maintained a constant temperature of 50° C. After waiting 15 minutes for thorough mixing, the humidified scCO.sub.2 was then flowed at various flow rates into a cold trap (see FIG. 2), which was maintained at −10° C. using a rock salt/ice water bath. The flow rate of the scCO.sub.2 was controlled using a back-pressure regulator (TESCOM, Elk River, Minn.). The mass of the trap was measured before and after each experiment using an analytical mass balance (Mettler Toldeo, Columbus, Ohio) to determine the amount of water dissolved in the scCO.sub.2 at each flow rate.

(22) The experiments were done at varying CO.sub.2 flow rates (1, 2.5, 5, 10, 15, and 20 mL/min, which can also be stated, respectively, as 0.994, 2.49, 4.97, 9.94, 14.9, and 19.9 g/min, since the density of CO.sub.2 at these conditions is 0.994 g/mL) but using the same total volume of CO.sub.2 (200 mL) each time to allow for fair comparisons between runs. The equilibrium solubility of water in supercritical CO.sub.2 at 50° C. and 2000 psi (p=0.665 g/mL) is 0.00837 mole fraction, so this level must be reached for the flow system to ensure that the CO.sub.2 is fully saturated with water and that water will not be extracted from the tissue sample during the decellularization process.

(23) For each flow rate, an “observed mole fraction” (x.sub.obs) was calculated based on the amount of water collected in the cold trap and the amount of CO.sub.2 used in the experiment. This x.sub.obs value should be equal to the equilibrium value if complete presaturation is achieved. The results obtained are shown in FIG. 3.

(24) The general trend of these results agrees with the hypothesis. At low flow rates, the observed mole fractions are around the equilibrium value, and as the flow rate is increased, the observed mole fraction decreases below this value. This occurs because the flow rate is too fast for adequate mixing to occur in the chamber before it flows into the cold trap.

(25) After confirming that the presaturator does achieve the equilibrium level of water in the scCO.sub.2, we moved to performing an experiment that involved contacting an actual test material with humidified scCO.sub.2 to determine if it would dehydrate that material compared to dry scCO.sub.2. The material chosen for these tests was poly(acrylic acid-co-acrylamide) potassium salt, a powder that swells to form a hydrogel in the presence of water. It has the capacity to absorb up to 200 mL of water per gram of powder. This model hydrogel was chosen for this experiment because, when hydrated, it is essentially composed entirely of water. Therefore, contacting the hydrogel with scCO.sub.2 allowed us to measure the extraction of water without having to worry about other variables, since this test avoids the possible complications of testing an animal tissue or other material.

(26) The tests were performed using the following apparatus, which is similar to the one used in the previous section, but with some changes, described in FIG. 4. The major addition is a second pressure cell, called the treatment chamber. It is a 10 mL view cell that is identical to the 25 mL mixing cell except for its smaller volume. The hydrogel sample, approximately 0.2 g, is fully swelled, blotted onto a nylon filter, and sealed inside the treatment chamber before treatment begins. A hand pump (HiP Pressure Generator 62-6-10, High Pressure Equipment Co., Erie, Pa/) is attached to the treatment chamber and used to maintain a slow, controlled depressurization of 50 psi/min after treatment. Finally, the cold trap is removed, and the CO.sub.2 is simply vented.

(27) Four sets of experiments were run: two control groups, where no water was loaded into the presaturation chamber, and two experimental groups, where the presaturator was utilized. All experiments were run at 2000 psi (13.79 MPa) and two different temperatures: 37° C. (ρ=0.769 g/mL at the given pressure) and 50° C. (ρ=0.665 g/mL). Each set consisted of four separate trials. For each trial, the mass of hydrogel used was approximately 0.2 g. Variation in hydrogel mass was accounted for by varying the treatment time slightly based on the mass of each sample; a treatment ratio of 30 minutes per 0.1 g of gel was used. This and other conditions used (including temperature, pressure, and depressurization rate) were chosen to be very similar to the conditions used by Sawada et al. to allow for easier comparison with their results.

(28) The data obtained are given in the tables below:

(29) TABLE-US-00001 TABLE 1 Treatment of Hydrogels with Supercritical CO.sub.2 at 37° C. Presaturated? Start Mass (g) End Mass (g) % Water Retained No 0.202 0.125 61.7 No 0.199 0.096 48.2 No 0.193 0.072 37.4 No 0.198 0.116 58.9 Dry Average: 51.5% ± 17.6% Yes 0.193 0.191 99.4 Yes 0.207 0.206 99.2 Yes 0.199 0.199 99.7 Yes 0.204 0.201 98.2 Presaturated Average: 99.1% ± 1.0% 

(30) TABLE-US-00002 TABLE 2 Treatment of Hydrogels with Supercritical CO.sub.2 at 50° C. Presaturated? Start Mass (g) End Mass (g) % Water Retained No 0.2031 0.0842 41.5% No 0.2016 0.1061 52.7% No 0.1952 0.1264 64.8% No 0.1919 0.0489 25.5% Dry Average: 46.1% ± 26.6% Yes 0.2079 0.2075 99.8% Yes 0.2077 0.2072 99.8% Yes 0.2108 0.2082 98.8% Yes 0.2032 0.1990 98.0% Presaturated Average: 99.1% ± 1.4% 
Statistical analysis was done for the 37° C. data series and the 50° C. data series, with very similar results found for both temperatures. At 37° C., the average water loss was 48.45%±17.64% for the control group and 0.87%±1.05% for the presaturated group. This difference is statistically significant using a Student's t-test with p<0.05. Similar results were obtained at 50° C., with an average water loss of 53.92%±26.58% for the control and 0.93%±1.42% for the presaturated group. This difference was also statistically significant for p<0.05. These results are shown in FIG. 5.

(31) Though the hydrogel results were promising, it was still necessary to perform experiments using actual tissues that could potentially be decellularized to determine the true viability of the presaturation method. Porcine aorta has been chosen to test since it was the tissue studied by Sawada et al., and there is currently a need for tissue-engineered heart valves and blood vessels.

(32) Porcine heart was obtained from a local slaughterhouse, and the aorta was isolated and surrounding fatty tissue was removed. The aortic tissue was cut into thin rectangles (approx. 3 cm×2 cm) and stored in phosphate buffered solution (PBS) at 4° C. for up to 48 hours until use. Each specimen was dried for 15 minutes under a light vacuum using filter paper and a Buchner funnel to remove free saline prior to weighing and treatment. Drying in a vacuum oven (37° C., 15 in Hg vacuum) was used as a negative control; changes in mass were recorded at 1, 2, 3, 6, and 24 hr. For CO2 treatments, a tissue section was loaded into the treatment chamber. The same experiments performed in section 3.2 were done with the porcine aorta tissue in place of the hydrogel (see FIG. 4). Tests were performed using dry (positive control) and presaturated (experimental) supercritical CO2 at 2000 psi (13.79 MPa) and 37° C. (p=0.769 g/mL) and with a scCO2 flow rate of 1 mL/min and depressurization rate of 50 psi/min (0.345 MPa/min). Treatment time was determined by using a treatment ratio of 60 min per 0.25 g tissue.

(33) After vacuum drying the tissue samples, a drying curve was generated for porcine aorta tissue (n=6), as shown in FIG. 6. The change in mass from 6 to 24 hours was found to be relatively insignificant, and therefore only data up to 6 hours is used in other figures. FIG. 7 shows FIG. 6 from 0-6 hours.

(34) Dimensionless mass is plotted on the y-axis to create a uniform drying curve for all samples tested. Vacuum drying of the native tissue removed over half of the initial mass in the first hour, with a continually more gradual decline in mass over the next five hours until reaching about 20% of the initial mass. This final mass makes sense, since the water content of porcine aorta is known to be approximately 80%.

(35) Results for positive control (n=4) and experimental (n=5) supercritical CO.sub.2 runs are given in Table 3, below.

(36) TABLE-US-00003 TABLE 3 Treatment of Porcine Aorta with Supercritical CO.sub.2 at 37° C. Presaturated? Native Mass (g) End Mass (g) % Mass Retained No 0.2534 0.1967 77.6 No 0.2205 0.1793 81.3 No 0.2425 0.1819 75.0 No 0.2068 0.1664 80.5 Dry Average: 78.6% ± 4.6% Yes 0.1811 0.1776 98.1 Yes 0.2148 0.2049 95.4 Yes 0.2569 0.2518 98.0 Yes 0.2466 0.241.1 97.8 Yes 0.2777 0.2696 97.1 Presaturated Average: 97.3% ± 1.4%

(37) The average losses in weight are 21.4%±4.6% for dry CO.sub.2 and 2.7%±1.4% for presaturated CO.sub.2. This difference is statistically significant for p<0.05, as shown in FIG. 8. It can be seen that using presaturated CO.sub.2 considerably reduces the amount of mass lost during treatment. However, there is still some mass loss using presaturated CO.sub.2, which gives rise to the possibility that a small amount of volatile substances other than water exist in the tissue, which needs to be investigated going forward.

(38) Presaturated scCO.sub.2 treatment was found to generally not cause extensive water removal. FIG. 9 shows where dry CO.sub.2 treatment lies on the tissue drying curve.

(39) A thermodynamic check can also be used to show that this comparison is reasonable. The solubility of water in supercritical CO.sub.2 is 0.00837 mole fraction. This value can be used to determine the maximum “extractable water” (EW) based on the moles of CO.sub.2 used in the treatment (which varies for each sample based on the treatment ratio). A ratio of actual EW to maximum EW can then be determined, as shown below:

(40) TABLE-US-00004 TABLE 4 Thermodynamic Check for Extractable Water Dry CO.sub.2 CO.sub.2 Max Act EW Sample # (mL) (mol) EW (g) EW (g) Ratio 1 60.816 1.083 0.163 0.0567 0.348 2 52.920 0.942 0.142 0.0412 0.290 3 58.200 1.036 0.156 0.0606 0.388 4 49.632 0.883 0.133 0.0404 0.304
The average ratio of actual to maximum EW is 33.05%, or about one-third. This value is reasonable because the conditions in the treatment chamber are not ideal for maximum extraction.

(41) The Cold Trap Experiments had the following sample calculations:

(42) $\mathrm{Conditions}\text{:}T=50°C.,P=2000\mathrm{psi}->{\rho }_{\mathrm{CO}2}=0.665g\text{/}\mathrm{mL};$${\rho }_{H2O}=0.944g\text{/}\mathrm{mL}$$V_{\mathrm{CO}2}=200\mathrm{mL},x_{\mathrm{obs},\mathrm{eq}}=0.00837$$\mathrm{Moles}{\mathrm{CO}}_2\text{:}{n}_{{\mathrm{CO}}_2}=200\mathrm{mL}*0.665\frac{g}{\mathrm{mL}}*\frac{1\mathrm{mol}}{44g}=3.028\mathrm{mol}$$\mathrm{Moles}{H}_{2}O\text{:}{n}_{H_{2}O}\left(\mathrm{from}\mathrm{expt}\right)*\frac{18g}{\mathrm{mol}}*\frac{1\mathrm{mL}}{0.994g}$$\mathrm{Observed}\mathrm{Mole}\mathrm{Fraction}\text{:}{x}_{\mathrm{obs}}=\frac{n_{H_{2}O}}{n_{{\mathrm{CO}}_2}}$

(43) Overall, the results from this section are very promising. The cold trap experiments show that the presaturation method used can effectively saturate scCO.sub.2 with water. The hydrogel experiments show that presaturating scCO.sub.2 with water prevents the humidified CO.sub.2 from extracting much, if any, water from the substance it contacts. It also shows that presaturation makes a significant difference in preventing this from happening, as the control tests with dry CO.sub.2 showed the same water extraction reported by Sawada et. al.

(44) The cold trap tests show that, even in a dynamic system, continuous presaturation can be achieved at low flow rates. The values of observed mole fraction at 1 mL/min and 2.5 mL/min were actually slightly greater than the known solubility of water in scCO2 at equilibrium. This likely occurred because of a light film that forms on the outside of the trap from being in the ice bath and is hard to completely remove prior to weighing the trap, thus increasing the value of the mass measurement just slightly. Regardless of the possible error caused by this, these results clearly show a trend of decreasing xobs as flow rate increases, which was expected based on our theoretical understanding of the process.

(45) The hydrogel experiments, in particular, are strong evidence that the presaturator works as intended. The positive controls show an expected dehydration of the hydrogels over time, caused by extraction of water by the dry CO.sub.2, resulting in about a 50% weight loss. The amount of water lost is slightly higher at 50° C., likely because the solubility of water in CO.sub.2 increases with temperature, which would cause more water to be extracted at 50° C. than at 37° C. On the other hand, the experimental group shows less than a one percent average weight loss at both temperatures, showing that the humidified CO.sub.2 extracts little or no water from the hydrogels. Analogous results were obtained when porcine aorta tissue was treated in place of the hydrogel.

(46) A novel method for treating tissues or other materials with presaturated supercritical CO.sub.2 has been presented, and its functionality has been verified by three different sets of experiments. From these results, we conclude that this method could potentially be useful for the decellularization of tissues, and that attempting decellularization is warranted based on these results.

(47) These and other modifications and variations to the present invention may be practiced by those of ordinary skill in the art, without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. In addition, it should be understood the aspects of the various embodiments may be interchanged both in whole or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention so further described in the appended claims.