Method and apparatus for changing one type of cell into another type of cell |
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申请号 | US13288801 | 申请日 | 2011-11-03 | 公开(公告)号 | US20120129224A1 | 公开(公告)日 | 2012-05-24 |
申请人 | Chauncey B. Sayre; | 发明人 | Chauncey B. Sayre; | ||||
摘要 | A method and apparatus converts host cells of a first type into cells of a second type when the host cells are placed in intimate contact with donor cells of the second type. Under predetermined conditions there is transport of a sufficient number of mRNA molecules from the donor cells into the host cells to reprogram the host cells into the second type. The host and donor cells may be subjected to while in intimate contact to a transporting force that enables the mRNA molecules of the donor cells to penetrate an outer membrane wall of host cells without damaging the membrane wall. The transporting force may include an electric field, a magnetic field, or a combined electric field and magnetic field. | ||||||
权利要求 | |||||||
说明书全文 | This application is a continuation patent application based on international patent application number PCT/US2010/33728, filed May 5, 2010, which claimed the benefit under 35 USC 119(e) of U.S. Provisional Patent Application No. 61/176,643, entitled “METHOD OF AND APPARATUS FOR CHANGING ONE TYPE OF CELL INTO ANOTHER TYPE OF CELL,” filed May 8, 2009. This related provisional patent application is incorporated herein by reference and made a part of this application. If any conflict arises between the disclosure of the invention in this PCT application and that in the related patent application, the disclosure in this PCT application shall govern. Moreover, any and all U.S. patents, U.S. patent applications, and other documents, hard copy or electronic, cited or referred to in this application are incorporated herein by reference and made a part of this application. The words “comprising,” “having,” “containing,” and “including,” and other forms thereof, are intended to be equivalent in meaning and be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. The words “substantially” and “essentially” are intended to be equivalent in meaning The word “cytoplasm” means the material of a cell between the nucleus of the cell and the outer membrane wall of the cell. The words “outer membrane wall” means the biological membrane separating the interior of a cell from the outside environment. The word “ova” are mature female reproductive cells that can divide to give rise to an embryo. The word “polyadenylation” means the addition of a poly(A) tail, a stretch of RNA where all the bases are adenines, onto an RNA molecule. The words “viral vector” mean a type of virus used in cancer therapy where a virus is changed in the laboratory and cannot cause disease. The words “differentiated cells” means cells derived by a process by which a less specialized cell becomes a more specialized cell type. The words “pluripotent cell” means a cell that is able to differentiate into many cell types. Different types of cells have different functions, for example, a beta cell of the pancreas secrets insulin and a cardiac cell of the atrium has a fluid action potential and contracts rhythmically. Converting one type of cell into another type of cell without any deleterious effects is a long sought after goal of molecular biologists. Also, such converted cells may be used in the medical treatment of numerous human and animal afflictions. But current methods for making such cells introduce into their structure substances that may be potentially harmful to a patient. For example, in converting differentiated cells into pluripotent cells, such techniques as chemicalporation, lypofection and plasmid utilization have been employed that introduce deoxyribonucleic acid (DNA) into host cells. This DNA has objectionable residual effects. Some have used viruses or viral vectors that again have objectionable residual effects. Examples of deleterious substances are foreign aliphatic compositions, metals, or chemicals that porate or otherwise damage the outer membrane wall of the cells. The processes used may also have negative effects. Ribonucleic acid (RNA) plays an important role in the growth of cells. RNA molecules are smaller than DNA molecules and usually are single-stranded, while DNA molecules are usually double-stranded. There are a wide variety of RNA molecules including messenger ribonucleic (mRNA) and micro ribonucleic (miRNA) molecules. The mRNA molecule encodes a chemical “blueprint” for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites within a cell of protein synthesis: the ribosomes. Here, a nucleic acid polymer is translated into a polymer of amino acids: a protein. The resulting proteins determine the type of living cell being produced. The miRNA regulate gene expression. miRNA molecules are partially complementary to one or more mRNA molecules, and their main function is to down-regulate gene expression. The mRNA and miRNA molecules include phosphate groups along their backbones that give these segments a negative charge. My method and apparatus of converting host cells into different cell types have one or more of the features depicted in the embodiments discussed in the section entitled “DETAILED DESCRIPTION OF SOME ILLUSTRATIVE EMBODIMENTS.” The claims that follow define my method and apparatus, distinguishing them from the prior art; however, without limiting the scope of my method and apparatus as expressed by these claims, in general terms, some, but not necessarily all, of their features are: One, in my method of converting host cells of a first type into cells of a second type the host cells are placed in intimate contact with donor cells of the second type. The intimate contact is under predetermined conditions that transport a sufficient number of mRNA molecules from the donor cells into the host cells to reprogram the host cells into the second type. The conditions may include the application of an electromagnetic force, using a media that enables the electromagnetic force to produce an electrophoresis effect that acts on the mRNA molecules from the donor cells to transport them through an outer cell membrane into the hosts cells, subjecting the host and donor cells while in intimate contact to a transporting force that enables the mRNA molecules of the donor cells to penetrate an outer membrane wall of host cells without damaging the membrane wall. The transporting force may include an electric field, a magnetic field, or a combined electric field and magnetic field. Two, my method of converting host cells of a first type into cells of a second type may include placing the host cells in intimate and direct contact with an activated ova in a matrix and subjecting the matrix to electrophoresis to transport an array of reprogramming substances being produced by the activated ova across an outer membrane of individual host cells into the host cell to reprogram the host cells. In one embodiment, my method of transferring a biologically active material to host cells comprises the steps of (a) placing the biologically active material on a first electrode of a pair of electrodes and placing on a second electrode of the pair of electrodes the host cells, and (b) positioning the electrodes closely together so there is a narrow gap between the electrodes, and (h) applying an electrical field across the closely spaced electrodes that has sufficient strength so that the biologically active material migrates across the gap and into the host cells on the second electrode. The gap does not exceed 50 microns, and may be, for example, substantially from 15 microns to 5 millimeters. The strength of the electric field may be, for example, substantially from 50 to 150 volts. A static direct electric field or a pulsating direct electric field may be applied across the electrodes. For example, the magnetic field may be applied across the gap concurrent with the application of the electric field, the direction of the magnetic field being substantially at a right angle to the direction of migration across the gap of the biologically active material. A gel may be disposed between the electrodes. The biologically active material may applied as a thin coating carried by the first electrode that has a thickness substantially from 0.015 to 4.0 millimeters and the host cells are applied as a thin coating carried by the second electrode that has a thickness substantially from 0.015 to 4.0 millimeters, the coatings being substantially planar and facing each other and the first electrode having a negative polarity and the second electrode having a positive polarity. The biologically active material may comprise activated ova that produces a mixture mRNA and miRNA in predetermined proportions. The biologically active material comprises a mixture of mRNA and miRNA molecules that are amplified in number from those originating from the activated ova, the amplified in number of mRNA and miRNA molecules being substantially in the same proportions as normally yielded by the activated ova. Three, my method also includes changing differentiate host cells into pluripotent cells, and may comprise the steps of (a) extracting mRNA and miRNA from an activated ova of a living organism when the ova is reprogramming its nucleus, (b) amplifying the number of molecules of mRNA and miRNA extracted from step (a), (c) processing the mRNA and miRNA from step (b) by polyadenylation, (d) placing the polyadenylated mixture of mRNA and miRNA on a negative electrode of a pair of electrodes and placing on a positive electrode of the pair of electrodes differentiate host cells to be transformed into the pluripotent cells, and (e) positioning the electrodes closely together so there is a narrow gap between the electrodes, and (f) applying an electrical field across the closely spaced electrodes that has sufficient strength so that the mRNA and miRNA migrate across the gap into the host cells on the second electrode to interact therewith to transform the host cells into the pluripotent cells. In my method changing differentiate host cells into pluripotent cells, subsequent to step (c) and prior to step (d), the mRNA and miRNA molecules are blended in predetermined proportions substantially in the same proportions as normally yielded by the activated ova. The ova may be chemically, electrically or mechanically stimulated to make the mRNA and miRNA. The mRNA and miRNA may be extracted using a centrifuge. The mRNA and miRNA may be charged negatively so they migrate to the positive electrode. In step (b) the mRNA and miRNA may be hydrolyzed in an aqueous solution, purified and freeze-dried prior to step (c). My method changing differentiate host cells into pluripotent cells may use a gel material within the gap, the gel material being selected from the group consisting of agarose, Matrigel, and acrilamide. The gel material may include an electrolyte, and my method may be conducted without the use of harmful substances that would impede clinical use. Four, my method includes separating the constituents of a mix of reprogramming substances including reprogramming proteins, mRNA and miRNA. It comprises the steps of (a) providing a gel within a light-transmitting container that has a closed end and an open end. (b) including makers within the container that identify by color separate zones along the length of the container corresponding to collection of separated constituents, and (c) under cryogenic temperature conditions, placing the mix in contact with the gel near the open end and centrifuging the container so the constituents separate and collect in different layers within the gel according to their mobility. My apparatus for transferring biologically active material to host cells is suitable for conducting my method. It comprises a support mounting member, a pair electrodes attached to the mounting member, and a power source. The electrodes have opposed planar surfaces facing each other. At least one electrode is moveably mounted so the electrodes have (a) a first, spaced apart position to enable the biologically active material to be placed on the planar surface of one electrode and the host cells to be placed on the planar surface of the other electrode, and (b) a second position where the electrodes are close together so there is a gap between the electrodes that does not exceed 50 microns. The power source applies an electrical field across the electrodes when in the second position that has sufficient strength so that the biologically active material migrates across the gap into the host cells on the second electrode. The electrodes may be substantially parallel plates. The apparatus may include means for applying a magnetic field across the gap concurrent with the application of the electric field. The direction of the magnetic field may be substantially at a right angle to the direction of migration across the gap of the biologically active material. These features are not listed in any rank order nor is this list intended to be exhaustive. Some embodiments of my method and apparatus are discussed in detail in connection with the accompanying drawing, which is for illustrative purposes only. This drawing includes the following figures (Figs.), with like numerals indicating like parts: My method enables biologically active material to be transferred into the nucleus of host cells in a manner to change the host cells into a new type of cell and avoid introducing harmful materials into the host cells or damaging the host cells. This transfer is achieved under the influence of an electromagnetic force, for example, an electrical field established between a pair of electrodes. The biologically active material, however, is essentially devoid of genetic substances such as DNA. A characteristic of the molecules of the biologically active material is their mobility in the electrical field as compared to DNA, or other unwanted and potentially harmful larger molecules that may be present. In one embodiment (direct transfer method) the biologically active material is the material being produced by donor cells and directly transferred to the host cells from the donor cells while in intimate contact. In another embodiment (indirect transfer method) the biologically active material is a mixture of the more mobile, smaller molecules derived from donor cells but the donor cells are not in contact with the host cells. The biologically active material may include (1) those proteins that reprogram the nucleus of host cells or (2) mRNA or (3) a mixture of mRNA and miRNA or (4) a mixture of mRNA, miRNA and the reprogramming proteins. In the direct transfer method the material being transported into the host cells comprises the mobile, smaller molecules: the mRNA and miRNA and some reprogramming proteins. For example, the biologically active material in the direct transfer method may be the material derived during the early stages after activation of an ova from a living organism. Activation may be achieved without fertilization or the ova may be fertilized. The ova is activated in conventional ways, for example, chemically, electrically or mechanically. The ova donor cells may be animal and the host cells human. The host cells are in intimate contact with the activated ova cells in a matrix that facilitates electrophoresis. While in such intimate contact, the matrix is subjected to electromagnetic conditions to induce electrophoresis within the matrix to transport an array of reprogramming substances being produced by the activated ova across the outer membrane wall of individual host cells. These reprogramming substances have the mobility that any undesirable material from the ova cells lack. Consequently, they separate from any undesirable material and pass through the outer membrane wall of the host cell to reprogram the host cells. Normally, the ova are unfertilized and may be from the same species as the host cells, but they do not necessary have to be, for example, bovine and porcine ova may be used to provide mRNA and miRNA and reprogramming proteins that are introduced into human host cells. In the indirect transfer method, the reprogramming substances (reprogramming proteins, mRNA and miRNA) are (1) an extracted mix separated from donor cells and transported into the host cells under the influence of an electromagnetic force or (2) reprogramming proteins, mRNA and miRNA from donor cells that are isolated and segregated and either individually, or in various combinations, are transported into the host cells under the influence of an electromagnetic force. The host cells of a first type are thus converted into cells of a second type depending on the chemical nature of these reprogramming substances. By placing the reprogramming substances in intimate contact with the host cells under suitable conditions, a sufficient number of the reprogramming substances migrate into the host cells to covert them into cells of a different type, such as differentiated cells into pluripotent cells. Such conditions include subjecting the reprogramming substances and host cells to electromagnetic energy while in intimate contact. In the indirect transfer method the biologically active material may be (1) substantially only the array of reprogramming proteins from the donor cells, (2) substantially only the mRNA from the donor cells, (3) substantially only a mixture of such donor cell mRNA and miRNA, or (4) a mix of all these from the donor cells. In any such mixture or mix the proportion of 7 constituents may be controlled so that the amount of each constituent is close to that which is present in the donor cells. In accordance with one feature of the indirect transfer method, the constituents of a starting mixture or mix derived from any source may be separated into individual constituents as subsequently discussed in greater detail. In both embodiments, after transfer of the biologically active material into the hosts cells, these cells are cultured for a sufficient time, for example several hours, for the host cells to change type. In both embodiments, the change in the host cells occurs without any significant adverse impact on the now converted host cells and without the use of detrimental materials or production procedures. Either embodiment of my method may be used to change differentiate host cells into pluripotent cells. For example, both embodiments of my method yields pluripotent cells characterized as having the potential to differentiate into a more diverse range of cells than pluripotent cells produced by known techniques. Potentially any viable cell type in a human or animal body may be a host cell or a donor cell, for example, spermatogonial, hESC, mesenchymal, hematopoetic, or liver cells. The reprogrammed cells produced by my method may be used clinically by medical practitioners depending on the nature of the disease being treated, and both embodiments may be useful for clinical purposes. (Here donor and host cells are usually of the same species). In both embodiments of my method, a media may be used that enables the electromagnetic energy to produce an electrophoresis effect that acts on the molecules of the biologically active material to transport them through an outer membrane wall of a host cell into the cytoplasm surrounding the nucleus of the host cell. This, for example, enables the mobile molecules to pass through the outer membrane wall of host cells without damaging these membrane walls. The transporting force may be an electric field, or a combined electric field and magnetic field. The magnetic field accelerates transfer. Freeze drying may be advantageously employed in both embodiments of my method. RNA for the host cells or the proteins for the host cells may be extracted from the cell type into which one wishes to convert the host cells. The donor cells are placed in a lyophilizer to dehydrate the donor cells and kept them at a very cool temperature to allow for freeze drying that maintains the shape and integrity of the molecules. When most of the water has been removed through freeze-drying, this material may now be coated onto a dielectric layer as subsequently discussed. The unique pluripotent cells produced by my method may subsequently be converted into differentiated cells by introducing into these cells mRNA or a mixture of mRNA and miRNA of selected differentiated cells. These newly constituted differentiated cells derived from pluripotent cells produced by my method are then injected into a patient. The selected differentiated cells providing the RNA do not have to be derived from the same species as the pluripotent cells; however, in some circumstances it may be best that they are of the same species, and mostly that they be both mammalian. Typical differentiated donor cells from which the RNA is extracted may be from potentially any viable cell type in a human body, including brain cells, retinal cells, spleen cells, heart cells, nerve cells, pancreatic cells, placental cells, ovarian cells, and epidermal cells. Consequently, donor's differentiate cells of one predetermined type may be used to make the pluripotent cells according to my method, and then these pluripotent cells may be the starting material to make differentiated cells of another type using RNA from selected differentiated cells from the same or a different donor. In one embodiment of my apparatus, the electric field is provided by a pair of electrodes, at least one electrode being movable with respect to the other electrode. The source of the biologically active material is placed on one electrode and the host cells are placed on the other electrode. An electrical insulating matrix is between the electrodes so, when they are in close proximity, they form an electrical capacitor structure that enables the biologically active material to migrate into the hosts cells upon application of a voltage across the electrodes. For example, the biologically active material may be applied as a thin coating to an exposed surface of a layer of dielectric insulting material that is mounted on a negative electrode and the host cells may be applied as a thin coating to an exposed surface of a layer of dielectric insulting material that is mounted on a positive electrode. The electrodes are pressed against each other with coatings of the biologically active material and the host cells in intimate contact. Then the voltage is applied across the electrodes at a sufficient level to cause migration of the biologically active material into the host cells without essentially any current flow between the electrodes. When the electrodes are in close proximity, there is only a narrow gap between them that is minimal but sufficient to suppress any current flow across the electrodes when voltage is applied. The electrical insulating matrix may be a gel-type substance that serves as a matrix and conduit for molecules of interest—specifically RNA. Suitable gel-type substances are, for example, agarose, Matrigel (a trademark of BD Biosciences, and acrilamide. An electrolyte, for example, a phosphate buffered saline (PBS) solution may be included in the biologically active material or in the matrix. The electrodes may be planar having surfaces that face each other. One or both electrodes may initially be mounted to move and are separated by a distance greater than, for example, 6 inches to allow formation of the dielectric layers and to apply to their respective electrodes the coatings of the biologically active material and host cells. Then, the electrodes are moved towards each other with the layers touching but the electrodes separated by a minimal distance to form a gap between the electrodes. An electrical field is now applied across the closely spaced electrodes that typically is substantially from 50 to 150 volts. The electric field is direct, and may be a continuous, static direct electric field or a pulsating direct electric field that intermittently applies the electric field across the electrodes. The gap between the electrodes typically does not exceed approximately 5 millimeters, and may be substantially from 15 microns to 5 millimeters. Optionally, a magnetic field may be applied across the gap concurrent with the application of the electric field. The direction of the magnetic field is substantially at a right angle to the direction of the electric field, which is substantially the direction of migration across the gap of the biologically active material. The thickness of each dielectric layer typically does not exceed 2 millimeters and typically is substantially from 0.05 to 4 millimeters. The thickness of each coating typically is substantially from 0.015 to 4.0 millimeters. The spacing between the electrodes and voltage applied across these electrodes is controlled so that essentially no current flows between the electrodes across the narrow gap. Rather, because of the negative polarity of the electrode carrying, for example, the mRNA and miRNA and the negative charged phosphate groups along the backbones of the mRNA and miRNA, the mRNA and miRNA molecules are repelled by this electrode when power is supplied, propelling the mRNA and miRNA molecules across the gap through the outer membrane wall into the interior of the host cells. As discussed above, the source of the mRNA and miRNA may be direct from donor cells, or may be derived from other sources. Reprogramming substances derived from sources other than directly from donor cells may comprise a mix of constituents including reprogramming proteins, mRNA and miRNA. This derived mix of mRNA and miRNA may be in substantially the same proportions produced by an ova in vivo within the body of the donor of the ova. The quantity of mRNA and miRNA in the derived mixture may be amplified in accordance with conventional procedures set forth in Section A. Moreover, in my indirect transfer method, though produced in vitro, the mRNA and miRNA reprogram the genome of the host cell in essentially the same way donor cells would in my direct transfer method. The mRNA and miRNA decompose much faster than DNA, and therefore have no objectionable residual effects. Consequently, if anything, for an economically viable manufacturing process, the effective life of the mRNA and miRNA should be extended. The mRNA and miRNA molecules are also subject to enzyme attack, and this should be minimized. In order to slow degradation of the amplified mRNA and miRNA molecules, these molecules may be processed by polyadenylation. This polyadenylated mixture may be placed on the negative electrode and the host cells to be transformed into the pluripotent cells may be placed on the positive electrode, and then the procedures discussed above are followed. In the Example 1 below, the mRNA and miRNA, and reprogramming proteins, are directly transferred from the ova to the differentiate host cells to be converted into pluripotent cells in accordance with my direct transfer method. For larger scale production of the pluripotent cells, my indirect method may be used were the number of molecules of the mRNA and miRNA used is greatly amplified in accordance with conventional methods. This provides a biologically active material that is a mixture of concentrated mRNA and miRNA in an aqueous solution that is purified and separated from other types of RNA and other unwanted chemicals. In the mixture of amplified mRNA and miRNA, the mRNA and miRNA molecules are in substantially the same proportions as produced by their complementary activated ova. The mRNA is able to be amplified 5 million-fold and the miRNA is able to be amplified 1,000-fold. The ratio of mRNA to miRNA varies depending on the application. Typically, after amplification the mRNA and miRNA are mixed according to their natural proportions occurring in the cell. Reprogramming substances provided without direct contact from donor cells may comprise a mix of constituents including organelles, reprogramming proteins, mRNA and miRNA. These constituents are separated and then the separated constituents are used individually, or combined in any suitable re-mixture or new combination, and used in the indirect transfer method discussed above. This separation method includes providing a separating gel within a light-transmitting container that has a closed end and an open end. The separating gel includes circumferential, color coded markers that are viewed through the light-transmitting container that identify by separate color zones along the length of the container the location where corresponding separated constituents collect within the separating gel under the influence of centrifugal forces. The separating gel forms under cryogenic temperature conditions, the mix of constituents is placed in contact therewith near the open end of the container and then spun, centrifuging the container so the constituents and kinetically separate and collect in different layers within the separating gel according to their mobility. Referring to As shown in The separating gel forms under cryogenic temperature conditions and may comprise use two fluorocarbons: perflurodecaline and perfluoro-proplamine that are used as a base for artificial blood. These fluorocarbons are very inert and do not interact with biological material. They are mixed in a 50:50 ratio, vigorously mixed and then placed in the tube, which is placed in dry ice or liquid nitrogen and kept at −79.2° C. to form the separating gel. The best accurate segmentation of the constituents of the mix occurs after centrifugation at −70° C. When the constituents are separated into the layers, the separating gel while in the semi-solid state is removed as a unity piece that is then sliced into sections to segregate the constituents. The following are examples of practicing my method.
Two electrodes at room temperature were taken and coated with Matrigel. The Matrigel was removed from −25 degree Celsius storage and placed on ice under a biosafety cabinet. The Matrigel was placed onto the facing surfaces of both electrodes at a concentration of 50 microliters (ul) per cm2, and both electrodes were incubated at 37 degrees Celsius for 30 minutes. A vial of approximately 6 million cryopreserved human hepatocytes was thawed according to the CellzDirect protocol as explained in Section B and applied as a coating to the Matrigel layer on the positive electrode. The porcine and bovine oocytes were transferred to the top of the Matrigel-coated electrode that had been covered with hepatocytes. Then the negative electrode was gently placed on top of the oocytes and pressure was applied. Next different electrical pulses were applied: one at 25 volts, one at 100 volts, one at 125 volts. These pulses were pulsed repeatedly and lasted for approximately one second intervals for a time period of approximately three minutes. The gold electrodes were removed and placed in a 35 mm dish containing media (Media First Day-Conception Technologies of San Diego, Calif.) that promotes oocyte growth in its early stages. The next day, the media was changed to Media Second Day (Conception Technologies). The third day, the cells were transferred to Media Day Three (Conception Technologies). On the fifth day, the cells were transferred to Media Day Five (Conception Technologies). The next day (Day 6), the excess media was siphoned off and 2 milliliters (ml) of phosphate buffered saline (PBS) were added to the dishes and vigorously mixed. This washing process was repeated three times. The cells were blocked with 6 ml of 4% normal Goat Serum in PBS and incubated at room temperature for 30 minutes. The excess goat serum was aspirated. The cells were then stained with five different sets of primary antibodies known to indicate pluripotency from Chemicon, part of Millipore in Temecula, Calif. These included: SSEA-3 (IgM), SSEA-4 (IgM), TRA-1-60 (IgM), TRA-1-81 (IgM), and Oct-4 (IgG). The antibodies were allowed to attach for two hours, and excess primary antibodies were then thoroughly washed off with 10 ml DPBS. Secondary antibodies from Millipore, including anti-SSEA-3, anti-SSEA-4, anti-TRA-1-60, anti-TRA-1-81, and anti-Oct-4 were added. The anti-SSEA-3, anti-SSEA-4, and anti-Oct-4 secondary antibodies were conjugated to FITC, which fluoresces green. The anti-TRA- and anti-TRA-1-81 were conjugated to Cy5, which fluoresces red. The antibodies were incubated with the cells for one hour at room temperature. The cells were washed with 10 mL DPBS to remove excess secondary antibodies. The cells were then separated from the 12.5 millimeters (mm) disc, spun down, and placed in a well, where a group of them fluoresced green, indicating the presence of any or all of three genes, including SSEA-3, SSEA-4 and Oct-4, which indicate pluripotency. This fluorescence was captured by camera.
To begin, six conductive gold electrodes were placed in individual wells of a 24 well plate, and then coated with Rat Tail Collagen Type I. 1500 microliters of collagen solution diluted to 50 micrograms/milliliters with 0.02 Normal acetic acid were prepared, and 250 microliters of this solution was placed in each well on top of the gold electrodes. The electrodes were incubated at room temperature for one hour allowing the collagen solution to harden on the surface of the electrode, and any excess remaining solution was aspirated. The electrodes were rinsed with PBS with calcium and magnesium to remove the acid. This rinse was repeated three times. Then 7.9 million human hepatocytes (CellzDirect) in a vial of were then thawed according to standard procedures in a 37 degree water bath. The cells were carefully washed with CHRM medium, centrifuged at 100× gravity for 10 minutes at room temperature, and resuspended in 2.5 ml of thawing/plating medium. 500 uL of the hepatocyte solution was placed on each electrode and the electrodes were incubated in a 37 degree 5% CO2 incubator for 6 hours. After this period, the thawing/plating medium was aspirated from each well, and replaced with incubation medium warmed to 37 degrees. The electrodes were returned to the incubator and left to recover overnight. After approximately eighteen hours, the gold electrodes were carefully removed from the wells with tweezers and placed in a 4 mm cuvette of the Gene Pulsar XL. The cuvette was filled with 100 microliters of the Total Human Placental RNA Solution and then the cuvette was placed in the chamber of the Gene Pulsar XL and three electrical pulses of 160V were applied for 1500 milliseconds at 500 microFarads, and infinite resistance. The hepatocytes plated on the gold electrodes were delicately removed from the cuvette with tweezers and returned to the individual wells. The cells were incubated at 37 degrees C. for six hours. After about 72 hours these hepatocytes became more translucent and histologically began to resemble human placental cells.
Six gold electrodes were coated and then placed in individual wells with CELLstart diluted 1:50 in Dulbecco's Phosphate Buffered Saline with calcium and magnesium. 160 uL of the diluted solution was placed in each well and incubated at 37 degrees Celsius, 5% CO2 for two hours. The excess CELLstart was aspirated. Next, we prepared 25 mL of the StemPro hESC SFM by combining 22.7 mL DMEM/F-12+Glutamax, 0.5 mL StemPro hESC SFM Growth Supplement, 1.8 mL Bovine Serum Albumin 25%, 20 uL FGF-basic and 45.5 uL 2-Mercaptoethanol. This hESC serum free medium was thoroughly mixed and warmed to 37 degrees Celsius. We then thawed a vial of approximately 2 million cryopreserved human embryonic stem cells in a 37 degree water bath. The cells were emptied into a 15 mL conical tube and warm hESC medium was added in a dropwise fashion until 10 mL of solution was reached. We spun the cells in a centrifuge for 4 minutes at 200×g, aspirated the supernatant and resuspended the cells in 2 mL hESC medium. 250 uL of this solution was applied to each of the six wells with a CELLstart-coated gold electrode. The hESCs were allowed to attach to the electrodes overnight in a 37 degree Celsius 5% incubator. The gold electrodes were then carefully loaded into a 4 mm cuvette with tweezers. 100 uL of Total Human Liver RNA was thawed in a 37 degree Celsius water bath and applied to the 4 mm cuvette. The cuvette was loaded into a Bio-Rad Gene Pulsar XL and we conducted three electrical pulses of 120V for 1500 ms at 500 uF, and infinite resistance. The hESCs plated on the gold electrodes were delicately removed from the cuvette with tweezers and returned to the individual wells. The plate was incubated at 37 degrees for six hours. In one of the liver trials, the hESC cells began to resemble liver cells. They became individualized rather than clumping together, and took on an elongated and brownish appearance compared to clear, round hESCs.
Polyadenylation
Reverse Transcription
Purification of First-Strand cDNA
Tailing of First-Strand cDNA
T7 Promotor Synthesis
In Vitro Transcription (Important to Use an Air Incubator—No Condensation Ok)
Purify senseRNA Amplification of mRNA Round One 1St Strand cDNA Synthesis
Round One 2nd Strand cDNA Synthesis
Round One In Vitro Transcription
Round One RNA Purification (Use Qiagen RNeasy MinElute Cleanup Kit)
Round Two 1st strand cDNA Synthesis
Round Two 2Nd Strand cDNA Synthesis
In Vitro Transcription of Aminoallyl-aRNA
Aminoallyl-aRNA Purification This step requires the Qiagen RNeasy MinElute Cleanup Kit
The above presents a description of the best mode I contemplate of carrying out my method and apparatus and of the manner and process of making and using them, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which they pertain to make and use my method and apparatus. My method and apparatus is, however, susceptible to modifications and alternate constructions from the illustrative embodiment discussed above which are fully equivalent. Consequently, it is not the intention to limit my method and apparatus to the particular embodiment disclosed. On the contrary, my intention is to cover all modifications and alternate constructions coming within the spirit and scope of my method and apparatus as generally expressed by the following claims, which particularly point out and distinctly claim the subject matter of my invention: |