WO2005079531A2

WO2005079531A2 - Method and system for eluting cells

Info

Publication number: WO2005079531A2
Application number: PCT/US2005/005490
Authority: WO
Inventors: James P. Landers; Jerome P. Ferrance; Jessica C. Voorhees; Kiev R. Blasier
Original assignee: University Of Virginia Patent Foundation
Priority date: 2004-02-19
Filing date: 2005-02-22
Publication date: 2005-09-01
Also published as: WO2005079531A3

Abstract

The present invention relates to method and system for eluting cells from materials for analysis and diagnostic examination by means of a swab, clothing items, or the like. The method includes incubating the cell-containing material in a solution that degrades the material while maintaining the cells or biological materials of interest, especially polynucleotides, such as DNA and/or RNA, intact. Preferably, the solution contains at least one enzyme for degrading the material. A system for practicing the method is also disclosed.

Description

METHOD AND SYSTEM FOR ELUTING CELLS

The present invention claims priority to U.S. Provisional Patent Application Serial No. 60/546,080, which is incorporated herein by reference. FIELD OF THE INVENTION The present invention relates to method and system for eluting cells from materials for analysis and diagnostic examination by means of a swab, clothing items, or the like. The method and systems of the present invention are applicable for use with microchip analysis.

BACKGROUND OF THE INVENTION In the medicinal, biological, or forensic sciences, cells, body fluids and microorganisms are commonly collected from humans, other living creatures, or from biological material deposited on surfaces for subsequent study. Typically, cells are removed from a body with a cotton swab and thereafter retained for further examination. In the case of forensic evidence, cells are also obtained from clothing, fabrics, filters, or swabs from the scene of the crime. Such cells may be placed on a slide for microscopic examination, on a culture medium for microbiological culture studies, or eluted in a solution for preserving the cells for subsequent analysis, such as DNA analysis, or for transport to a laboratory. Procedures are routinely performed to remove and study cells from such body cavities as the nasal, throat, anal, vaginal, cervical, and urethral cavities as well as from surgical wounds and topical skin sites using a cotton swab. The cells taken with a cotton swab or collected from clothing may only provide a minimal amount of material for proper analysis. Many cells adhere to the surface of the swab or clothing and especially between the cotton fibers. Often, fifty percent or more of the total specimen is left in the cotton swab and is lost for the diagnostic evaluation unless recovered by additional complicated washing processes and then converted into a suspension. Currently methods for eluting cells from cotton fibers, similar to the ones used by the FBI and state agencies, include the use of proteinase K or other cell lysis buffers. Cotton swab samples are incubated in solution in a water bath long enough for the cellular material to be lysed or desorbed. The samples are then centrifuged to collect the desorbed cells and separate it from the lysed cellular material in a microcentrifuge tube. A drawback of this method is that not all the cells are released from the cotton; large amounts of cells remain bound to the cellulose fibers. This can be especially problematic in situations where there is a limited amount of cells present, such as for example, samples from a crime scene. In addition to cellulosic swabs or material, cellulosic or nitrocellulosic filters can be used for collection of cells or biological materials from filtration of air or water samples. This method of collection can be utilized in the sample for determination of the release of biowarefare agents through aerosol or water delivery methods. Removal of material from these filters should also be enhanced through the use of enzymes to degrade which degrade the crystal structure of the fiber material. Enhanced removal greatly increases the material recovered for subsequent analysis and identification of organisms, toxins, or other agents. Therefore, there remains a need for a method for eluting cells from cellulosic materials that is capable of high efficiency and yield, while using a smaller elution volume. SUMMARY OF THE INVENTION An object of the present invention provides a method for eluting cells or biological materials, such as sperm cells, DNA, RNA, and proteins, from materials, such as cellulose, leather, and the like. The method includes incubating the cell- containing material in a solution that degrades the material while maintaining the cells or biological materials of interest, especially polynucleotides, such as DNA and/or RNA, intact. Preferably, the solution contains at least one enzyme for degrading the material. For cellulosic material, the enzyme can be cellobiohydrolase, endoglucanase, cellulase, hemicellulase, β-glucosidase, or combinations thereof. The

benefits of this method include: 1) higher extraction efficiencies and yields; 2) increased speed for extraction and elution; 3) elution of more cells or biological materials in a smaller elution volume; and 4) smaller samples required for analysis. Another object of the present invention provides a system for eluting cells or biological materials for use with a microchip microfluidic analysis system. The system includes a block for holding a sample tube, where the block contains a puncture blade and a channel for carrying the concentrated cells or biological materials to the microchip for analysis. The present invention details a separate system for incubation and transfer of material into the microchip, however, the sample could also be directly placed into a reservoir on the microchip with the enzymatic digestion and incubation performed directly on the microchip. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a scanning electron micrograph showing the fixing of sperm cells to cotton fiber crystals; Figure 2 is a bar graph comparing sperm cell elution from cotton using the present method and that of the prior art; Figure 3 is a bar graph comparing different elution solutions; Figure 4 is a graph comparing sperm cell elution using the present enzyme solution and no enzyme; Figure 5 is graphs showing sperm elution efficiency as a function of cellulase concentration. The x-axis shows concentration of cellulase from A. niger in citrate buffer) a) samples dried for 2 days, b) samples dried for 10 weeks; Figure 6 is a graph comparing the effectiveness of cellulase from three different organisms; Figure 7 is a graph showing the synergistic effect of an appropriate buffer and cellulase. MDEB - modified differential extraction buffer consisting of Tris, Sarkosyl, NaCl, and EDTA; Figure 8 is a graph showing the effect of Sarkosyl on epithelial cells elution. Differential extraction buffer is known to lyse epithelial cells, so there was none recovered intact with this buffer as expected. MDBE-1 is modified differential extraction buffer- 1 without proteinase K or Sarkosyl (Tris, NaCl, EDTA); MDBE-2 is modified differential extraction buffer-2, with no proteinase K included (Tris, NaCl, EDTA, Sarkosyl). Inclusion of the enzyme showed enhancement in the number of intact epithelial cells released from the swabs in citrate buffer, but not in MDEB-2; and Figure 9 is a diagram of the present system for use with the μTAS. Left: angled side-on view showing how the SIT would be inserted, and how the interface connects to the microchip. Right: side view showing how the blade would slit the SIT allowing for the liquid to be moved out through the interface drain to the microchip reservoir by spinning.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Cellulose is represented by the general formula (C₆H₁₀O₅)_n. Materials containing cellulose as the main component are exemplified by woods such as pines, cedars, beeches and poplars; stalks and basts such as hemps, paper bushes, rice straws, bagasse and chaff; seed downs such as cotton; old papers such as newspapers, magazines and corrugated cardboard waste papers; and other fibrous materials, such as, pulps, cellulose powder and the like. A cellulose molecule has a structure in which D-glucopyranose is connected though β-1,4 bonds and has no side chain. Thus, cellulose is composed of a polymer of glucose. Cellulose generally forms very tightly packed crystals via its hydrogen bonding capability. Elution of cells or biological materials from cellulosic materials is fraught with low yield and inefficiency because the cells or biological materials tend to stick to the cellulose crystal. For example, the scanning electron micrograph of Figure 1 shows the heads and tails of the sperm cells affixed to the polysaccharide cellulose strands. The present inventors have discovered that degrading the cellulose to glucose greatly enhances the efficiency and yield of cell release from cellulosic materials. Therefore, an embodiment of the present invention relates to a method of eluting cells or biological materials from cellulosic materials, such as cotton. The cell-containing cellulosic material in incubated in a solution that degrades the cellulosic fibers while maintaining the cells or biological materials, especially polynucleotides, such as DNA and or RNA, intact. The solution preferably contains at least one enzyme that can be cellobiohydrolase, endoglucanase, cellulase, hemicellulase, β-glucosidase, or combinations thereof. The enzyme concentration should be about 1 μg/mL to 1 mg/mL, preferably about 50-500 μg/mL. The optimal concentration of enzyme depends on many factors one of which is the age of the sample (see Figure 5). Generally, the older the age of the sample the higher the enzyme concentration required. A second factor is the activity of the enzyme preparation as illustrated by the greater number of cells released at the same concentration with the cellulase enzymes prepared from Trichoderma verdi (Figure 6). Additional enzymes or buffer components that yield a synergistic effect with the enzymes are also appropriate for the present invention. The enzymes are able to degrade cellulose and hemicellulose present in cotton and penetrate the strong crystals that form cellulose to release trapped cells. Further, because the enzymes have specificity for cellulose, which is not present in the cells of interest, the cells remain intact and are not affected by the elution process. Preferably the enzymes are dissolved in a buffered solution, preferably, citrate buffer at a pH appropriate to optimize the enzyme activity, usually about 10 mM citrate buffer at a pH of about 5.0-5.5. The buffer generally involves dissolving tri- sodium citrate in water and adjusting to the appropriate pH with acetic acid or sodium hydroxide. Applicant has also discovered that the addition of Sarcosyl (N- lauroylsarcosine or sodium lauroyl sarcosinate) improves the elution of cells and biological materials from the cellulosic material (see Figures 7-8); therefore, any buffer solution used preferably includes Sarcosyl. Cellulases are enzymes that hydrolyze the β-D-glucosidic linkages in celluloses. Cellulolytic enzymes have been traditionally divided into three major classes: endoglucanases, cellobiohydrolases (exoglucanases), and β-glucosidases. Cellulases are known to be produced by a large number of bacteria, yeasts and fungi. Commercially prepared cellulases are, in most cases, optimized mixtures of exoglucanase, cellobiohydrolases, and β-glucosidase. For example, Aspergillus niger cellulase that has been used for studies depicted in Figures 2-4 consists of a mixture of amylase, protease, and endoglucase, with exoglucanase and β-gluςosidase present in trace amounts. Cellulases produced by Trichoderma viride are most preferred for the present invention. Figure 6 compares effectiveness of cellulases from three different organisms, A. niger, Trichoderma reesei, and T. viride. It appears that all cellulases are more effective in eluting sperm cells than no enzyme at all; however, cellulase from T. viride is most effective. If the cellulase does not contain all three of exoglucanase, cellobiohydrolase, and β-glucosidase, each of the enzymes may also be added to the enzyme mixture individually. Cellobiohydrolase (exoglucanase) hydrolyzes a glucoside bond in a polysaccharide or an oligosaccharide consisting of D-glucose bonded through β- 1 ,4 bonds to release cellobiose, a disaccharide in which D-glucose is bonded through a β- 1,4 bond, but does not hydrolyze the glucoside bond in cellobiose. Cellobiohydrolases have been found to be produced by various types of organisms, such as plants and microorganisms, either naturally or recombinantly, all of which are appropriate for the present invention, depending on the specificity of the particular cellobiohydrolases. Endoglucanase hydrolyzes β-l,4-D-glycosidic linkages present in any cellulosic material. Endoglucanases have been found to be produced by various types of organisms, such as plants and microorganisms, either naturally or recombinantly, all of which are appropriate for the present invention depending on the specificity of the particular endoglucanase. The endoglucanase produced by Trichoderma reesie is preferred for the present invention. β-glucosidase catalyzes the hydrolysis of glycosidic linkages in aryl and alkyl β-glucosides and cellobiose and occurs ubiquitously in plants, fungi, animals and bacteria. Any β-glucosidase, natural or recombinant, is appropriate for the practice of the present invention. Hemicellulase catalyzes the degradation and/or modification of hemicelluloses, including xylanase, mannanase, xylosidase, mannosidase, glucosidase, arabinosidase, glururonidase, and galactosidase. Hemicellulases are produced naturally by various microorganisms, including bacteria and fungi, and also occur in some animals and in numerous plants. Among the microorganisms that produce such mannanases are species of Aeromonas, Aspergillus, Streptomyces, Rhodococcus and Bacillus, all of which are appropriate for the present invention. The present invention can also be practiced with recombinantly produced hemicellulase. Because cotton contains both cellulose and hemicellulose, a mixture of enzymes is best used to penetrate into the crystals forming the cotton and degrade it to release the cells or biological materials adhered thereon. Although cellulase, cellobiohydrolase, endoglucanase, β-glucosidase, and

hemicellulase are specifically disclosed herein, other enzymes or substances that degrade cellulosic materials are appropriate for the present invention, provided that the enzymes or substances do not also degrade the cells or biological materials of interest. Referring to Figures 2-4, which clearly show that the amount of sperm extracted from cotton can be improved at least two folds by using at least one cellulase enzyme by incubating the cotton in the enzyme solution. Further, Figure 3 clearly shows that more sperm cells can be eluted from cotton using a mixture of all five enzymes than cellulase alone or without any enzyme. Moreover, according to Figure 4, the use of the enzyme mixture of the present achieves twenty-fold increase in the number of sperm cells eluted from the cotton after 8 hours of incubation. Another embodiment of the present invention relates to a system for using the elution method described above with a microchip total analysis system (μTAS). Referring to Figure 9, the system contains a block (6) containing a hole (2) for holding a sample tube (4), preferably a centrifuge tube, therein. The hole (2) is preferably shaped so that the sample tube (4) fits exactly into the hole at an angle, preferably a 45 degree angle that is commonly used by commercially available centrifuges. The hole further contains a puncture blade (12) for punching a hole in the bottom of the sample tube (4). Preferably, the hole is punched when the tube (4) is inserted into the hole. Alternatively, the blade (12) is centrifugally activated to puncture the tube when the cells or biological materials are spun to the bottom of the sample tube (4). In another alternative, the blade (12) is manually actuated so that a user can manually puncture the tube (4) by pressing an actuator, such as a button. Puncture of the sample tube (4) allows the cells or biological materials collected at the bottom of the tube (4) to be removed and drain into the drain channel (8). The drain channel (8) preferably connects to a μTAS chip (10) for analysis of the cells or biological materials. The microchip (10) can be connected to the drain channel (8) through a tubing system or directly as illustrated in Figure 5. In the direct connection, the block (6) sits directly above the microchip (10) such that the exit of the drain channel sits directly above a sample reservoir on the microchip (10) so that the cells or biological materials are collected directly in the reservoir. Any μTAS or microfluidic systems in the art can be used with the present system. Exemplary systems are disclosed, for example, in U.S. Patent Publication Nos. 20050003411 to Chiu et al.; 20030092016 to Wiggins et al; 20020181837 to Wang et al; U.S. Patent Nos. 6,630,353 and 6,306,659 to Parce et al; 6,274,089 and 6,551,836 to Chow et al.; 5,427,946 and 5,744,366 to Kricka et al.; 5,928,880 and 6,551,841 to Wilding et al; the disclosures of which are incorporated herein by reference. In use, a cell-containing cellulosic material is incubated with the enzyme solution describe above for a predetermined amount of time. The material can be incubated in the sample tube (4) or eventually transferred to the sample tube (4) after the incubation period. The sample tube (4) is then placed in to the hole (2) of the block (6) which is then punctured and centrifuged for an amount of time determined by the cells or biological materials to be collected. The puncture in the sample tube (4) allows the cells or biological materials to drain into the drain channel (8) and eventually into the microchip (10) for analysis. The method of the present invention is not limited to the system described and illustrated above and in Figure 9. In another embodiment, direct addition of the sample to a reservoir on a microfluidic analysis system (microchip), with the incubation performed directly on the microchip can also be accomplished. This is limited by the size of the sample and the volume of elution buffer. Incubation on the microchip at an elevated temperature can be performed using technology currently available for heating on microchip. In this embodiment, no transfer mechanism is required as the eluted material is already contained on the device. Although the discussion above is drawn to cellulosic material, in another embodiment, the principle of the present invention can be expanded to other materials, such as leather. In the case of leather, elution of cells or other biological materials requires the use of enzymes that break down the leather matrix, namely proteases, such as trypsin and chymotrypsin. When other matrix materials are considered, the enzyme or enzyme mixture should be tailored to degrade that particular material. Importantly, however, the enzyme or enzyme mixture should not affect the biological material(s) being eluted from the material. In collecting samples from non-cellulosic material, the material can be swabbed with a cotton swab and the cells or biological materials can be obtained from the swab as described above. Although certain presently preferred embodiments of the invention have been specifically described herein, it will be apparent to those skilled in the art to which the invention pertains that variations and modifications of the various embodiments shown and described herein may be made without departing from the spirit and scope of the invention. Accordingly, it is intended that the invention be limited only to the extent required by the appended claims and the applicable rules of law.

Claims

What is claimed is

1. A method for obtaining cells or biological materials from a material comprising the step of incubating the material with a enzyme mixture containing at least one enzyme capable of degrading the material.

2. The method of claim 1, wherein the material is a cellulosic material.

3. The method of claim 2, wherein the at least one enzyme is selected from the group consisting of cellobiohydrolase, endoglucanase, cellulase, hemicellulase, and β- glucosidase.

4. The method of claim 2, wherein the cellulosic material is cotton.

5. The method of claim 2, wherein the cells are sperm cells.

6. The method of claim 2, wherein the enzyme mixture is dissolved in citrate buffer.

7. The method of claim 2, wherein the at least one enzyme is selected from the group consisting of cellobiohydrolase, endoglucanase, cellulase, hemicellulase, and β-

glucosidase.

8. The method of claim 7, wherein the enzyme is present at a concentration of about 1 μg/mL to about 1 mg/mL.

9. The method of claim 1, further comprising centrifuging the incubation mixture.

10. The method of claim 1, wherein the material is leather.

11. The method of claim 10, wherein the at least one enzyme is a protease.

12. The method of claim 1, wherein the enzyme mixture does not degrade the cells or biological materials.

13. An apparatus for the elution and analysis of cells or biological materials from a material comprising a block having a hole for holding a sample tube, a puncture blade, and a drain channel at the bottom of the hole.

14. The apparatus of claim 13, wherein the drain channel is capable of carrying the cells or biological materials eluted from the material to a microchip microfluidic system.

15. The apparatus of claim 13 , wherein the puncture blade is manually actuated.

16. The apparatus of claim 13, further comprising a microchip total analysis system (μTAS), wherein the block sits on top of the μTAS such that the drain channel is directly above a sample reservoir on the μTAS.

17. The apparatus of claim 13, wherein the material is a cellulosic material.

18. The apparatus of claim 13, wherein the material is leather.