Source: http://www.google.com/patents/US7155288?dq=7181427
Timestamp: 2016-06-27 00:58:49
Document Index: 279271174

Matched Legal Cases: ['application No. 60', 'art 56', 'art 56', 'art 56', 'art.\n81', 'art 2']

Patent US7155288 - Method and system for myocardial infarction repair - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsAn implantable system is provided that includes: a cell repopulation source comprising genetic material, undifferentiated and/or differentiated contractile cells, or a combination thereof capable of forming new contractile tissue in and/or near an infarct zone of a patient's myocardium; and an electrical...http://www.google.com/patents/US7155288?utm_source=gb-gplus-sharePatent US7155288 - Method and system for myocardial infarction repairAdvanced Patent SearchPublication numberUS7155288 B2Publication typeGrantApplication numberUS 11/300,176Publication dateDec 26, 2006Filing dateDec 14, 2005Priority dateNov 7, 1997Fee statusLapsedAlso published asUS7031775, US20040087019, US20060095089Publication number11300176, 300176, US 7155288 B2, US 7155288B2, US-B2-7155288, US7155288 B2, US7155288B2InventorsOrhan Soykan, Maura G. DonovanOriginal AssigneeMedtronic, IncExport CitationBiBTeX, EndNote, RefManPatent Citations (45), Non-Patent Citations (54), Referenced by (23), Classifications (21), Legal Events (5) External Links: USPTO, USPTO Assignment, EspacenetMethod and system for myocardial infarction repair
US 7155288 B2Abstract
(a) a cell repopulation source of stem cells for a patient's myocardium; and
(b) an electrical stimulation device for electrically stimulating the new contractile tissue in and/or near the infarct zone of the patient's myocardium, wherein the electrical stimulation device provides burst stimulation.
2. A method of repairing the myocardium of a patient, the method comprising:
(i) a cell repopulation source comprising genetic material, stem cells, or a combination thereof, capable of forming new contractile tissue in and/or near an infarct zone of a patient's myocardium; and
(ii) an electrical stimulation device for electrically stimulating the new contractile tissue in and/or near the damaaed or diseased myocardial tissue;
(b) implanting the cell repopulation source into and/or near damaged or diseased myocardial tissue of a patient;
(c) allowing sufficient time for new contractile tissue to form from the cell repopulation source; and
(d) electrically stimulating the new contractile tissue.
3. The method of claim 2 wherein the electrical stimulation device comprises an electrical stimulation device with electrodes; wherein the electrodes are implanted to stimulate said new contractile tissue.
4. The method of claim 2 wherein the cell repopulation source is delivered through a catheter.
5. The method of claim 2 wherein the cell repopulation source comprises autologous cells.
6. The method of claim 2 wherein the cell repopulation source comprises mesenchymal stem cells.
7. The method of claim 2 wherein the step of implanting the cell repopulation source into and/or near damaged or diseased myocardial tissue the cells are implanted into the infarct zone of the myocardiumof a patient.
8. The method of claim 2 wherein the electrical stimulation device is implantable and is in the form of a capsule having electrodes incorporated therein.
9. The method of claim 8 wherein the electrical stimulation device is a carrier for the cell repopulation source.
(a) a cell repopulation source of stem cells capable of forming new contractile tissue in and/or near damaged or diseased myocardial tissue; and
(b) an electrical stimulation device for electrically stimulating the new contractile tissue formed in the myocardial tissue.
11. The implantable system of claim 1 wherein the cell repopulation source of stem cells is a mesenchymal stem cell.
12. The implantable system of claim 10 wherein the cell repopulation source of stem cells comprise autologous cells.
13. The implantable system of claim 10 wherein the cell repopulation source further comprises a polymeric matrix.
14. The implantable system of claim 10 wherein the electrical stimulation device provides burst stimulation.
15. The implantable system of claim 10 wherein the electrical stimulation device provides pulse stimulation.
16. The implantable system of claim 10 wherein the cell repopulation source of stem cells comprise allogenic cells.
17. The implantable system of claim 10 wherein the cell repopulation source is associated with a carrier.
18. The implantable system of claim 17 wherein the cell repopulation source is coated on a carrier.
19. The implantable system of claim 1 wherein the electrical stimulation device further comprises an electrical stimulation device having two electrodes connected thereto.
20. The implantable system of claim 19 wherein the is electrical stimulation device implantable and is in the form of a capsule having electrodes incorporated therein.
21. The implantable system of claim 20 wherein the electrical stimulation device is a carrier for the cell repopulation source.
22. The implantable system of claim 10 wherein the cell repopulation source comprises genetic material.
23. The implantable system of claim 22 wherein the genetic material comprises plasmid DNA.
24. The implantable system of claim 22 wherein the genetic material comprises a delivery vehicle comprising a nucleic acid molecule.
25. The implantable system of claim 24 wherein the nucleic acid molecule encodes a myogenic determination gene.
26. The implantable system of claim 24 wherein the delivery vehicle comprises a viral expression vector.
27. The implantable system of claim 24 wherein the delivery vehicle comprises liposomes.
28. The implantable system of claim 24 wherein the nucleic acid molecule encodes VEGF.
29. The implantable system of claim 24 wherein the nucleic acid molecule encodes aFGF.
CROSS-REFERENCE TO RELATED TECHNOLOGY This application is a continuation of patent application Ser. No. 10/692,878 filed Oct. 24, 2003, now U.S. Pat. No. 7,031,775, which is a continuation of patent application Ser. No. 09/706,531 filed Nov. 3, 2000, now U.S. Pat. No. 6,671,558, which is a continuation-in-part of patent application Ser. No. 09/654,185 filed Sep. 1, 2000, now U.S. Pat. No. 6,775,574, which is a continuation of patent application Ser. No. 09/145,743 filed Sep. 2, 1998, now U.S. Pat. No. 6,151,525 which claims priority to provisional application No. 60/064,703 filed on Nov. 7, 1997. This application is related to patent application Ser. No. 10/824,011 filed Apr. 14, 2004 entitled “Method and System for Myocardial Infarction Repair” which is incorporated herein by reference.
FIELD OF THE INVENTION The present invention relates to methods and implantable systems to reverse damage to heart muscle following myocardial infarction and more generally in and/or near damaged or diseased myocardial tissue. Specifically, this involves the repopulation of the damaged or diseased myocardium with undifferentiated or differentiated contractile cells, which additionally may be formed in situ through the use of genetic engineering techniques, and augmentation with electrical stimulation.
BACKGROUND OF THE INVENTION Coronary Artery Disease (CAD) affects 1.5 million people in the USA annually. About 10% of these patients die within the first year and about 900,000 suffer from acute myocardial infarction. During CAD, formation of plaques under the endothelial tissue narrows the lumen of the coronary artery and increases its resistance to blood flow, thereby reducing the O2 supply. Injury to the myocardium (i.e., the middle and thickest layer of the heart wall, composed of cardiac muscle) fed by the coronary artery begins to become irreversible within 0.5–1.5 hours and is complete after 6–12 hours, resulting in a condition called acute myocardial infarction (AMI) or simply myocardial infarction (MI).
Those who survive AMI have a 4–6 times higher risk of developing heart failure. Current and proposed treatments for those who survive AMI focus on pharmacological approaches and surgical intervention. For example, angioplasty, with and without stents, is a well known technique for reducing stenosis. Most treatments are designed to achieve reperfusion and minimize ventricular damage. However, none of the current or proposed therapies address myocardial necrosis (i.e., degradation and death of the cells of the heart muscle). Because cardiac cells do not divide to repopulate the damaged or diseased region, this region will fill with connective tissue produced by invading fibroblasts. Fibroblasts produce extracellular matrix components of which collagen is the most abundant. Neither the fibroblasts themselves nor the connective tissue they form are contractile. Thus, molecular and cellular cardiomyoplasty research has evolved to directly address myocardial necrosis.
Cellular cardiomyoplasty involves transplanting cells, rather than organs, into the damaged or diseased myocardium with the goal of restoring its contractile function. Research in the area of cellular cardiomyoplasty is reviewed in Cellular Cardiomyoplasty: Myocardial Repair with Cell Implantation, ed. Kao and Chiu, Landes Bioscience (1997), particularly Chapters 5 and 8. For example, Koh et al., J. Clinical Invest., 96, 2034–2042 (1995), grafted cells from AT-1 cardiac tumor cell line to canines, but found uncontrolled growth. Robinson et al., Cell Transplantation, 5, 77–91 (1996), grafted cells from C2C12 skeletal muscle cell line to mouse ventricles. Although these approaches produced intriguing research studies, cells from established cell lines are typically rejected from the human recipient. Li et al., Annals of Thoracic Surgery, 62, 654–661 (1996), delivered fetal cardiomyocytes to adult mouse hearts. They found improved systolic pressures and noticed that the presence of these cells prevented remodeling after the infarction. Although their results showed the efficacy of transplanted cell technology, this approach would not likely be effective in clinical medicine since the syngeneic fetal cardiac tissue will not be available for human patients. Chiu et al., Ann. Thorac. Surg., 60, 12–18 (1995) performed direct injection of cultured skeletal myoblasts to canine ventricles and found that well developed muscle tissue could be seen. This method, however, is highly invasive, which compromises its feasibility on human MI patients.
This concept has been well-developed in vitro. For example, Tam et al., J. Thoracic and Cardiovascular Surgery, 918–924 (1995), used MyoD expressing retrovirus in vitro for fibroblast to myoblast conversion. However, its viability has not been demonstrated in vivo. For example, Klug et al., J. Amer. Physiol. Society, 1913–1921 (1995), used SV40 in vivo and succeeded in replicating the nucleus and DNA, but not the cardiomyocytes themselves. Also, Leor et al., J. Molecular and Cellular Cardiology, 28, 2057–2067 (1996), reported the in situ generation of new contractile tissue using gene delivery techniques.
Acsadi et al, The New Biol., 3, 71–81 (1991).
Barr et al., Gene Ther., 1, 51–58 (1994).
Cell Implantation”, Ann. Thorac. Surg., 60, 12–18 (1995).
French et al., Circulation, 90, 2414–2424 (1994).
Gal et al., Lab. Invest., 68, 18–25 (1993).
Johns, J. Clin. Invest., 96, 1152–1158 (1995).
Klug et al., J. Amer. Physiol. Society, 1913–1921 (1995).
Koh et al., J. Clinical Invest., 96, 2034–2042 (1995).
Leor et al., J. Molecular and Cellular Cardiology, 28, 2057–2067 (1996)
Li et al., Annals of Thorasic Surgery, 60, 654–661 (1996).
Murry et al., J. Clin. Invest., 98, 2209–2217 (1196)
Parmacek et al, J. Biol. Chem., 265, 15970–15976 (1990).
Parmacek et al., Mol. Cell. Biol., 12, 1967–1976 (1992).
Robinson et al., Cell Transplantation, 5, 77–91 (1996).
Tam et al., J. Thorasic and Cardiovascular Surgery, 918–924 (1995).
Proceedings of the Association of American Physicians, 109, 245–253 (1997).
von Recumin et al., Biomaterials, 12, 385–389, “Texturing of Polymer Surfaces
von Recumin et al., Biomaterials, 13, 1059–1069, “Macrophage Response to
von Recumin et al., Journal of Biomedical Materials Research, 27, 1553–1557,
SUMMARY OF THE INVENTION The present invention also provides methods and implantable systems that reverse the damage to necrotic heart muscle following myocardial infarction or in and/or damaged or diseased myocardial tissue. Specifically, this involves combining a method of supplying a source of a repopulating agent with a stimulation device. More specifically, this involves the repopulation of the damaged or diseased myocardium with undifferentiated or differentiated contractile cells and augmentation of the newly formed tissue with electrical stimulation to cause the newly formed tissue to contract in synchrony with the heart to improve the cardiac function.
The cell repopulation source may comprise undifferentiated contractile cells, such as skeletal muscle satellite cells, myoblasts, stem or mesenchymal cells and the like, or differentiated cardiac or skeletal cells, such as cardiomyocytes, myotubes and muscle fiber cells, and the like. The implanted cells may be autologous muscle cells, allogenic muscle cells or xenogenic muscle cells,
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention comprises (a) a cell repopulation source capable of forming new contractile tissue in and/or near damaged or diseased myocardial tissue. The cell repopulation source may be implanted into a patient's myocardium, preferably wherer the myocardium has been damaged or diseased, such as where the tissue is after a myocardial infarction. The repopulation source may be delivered directly to the myocardial tissue, such as in an infracted tissue area, by a catheter or more manually by a syringe.
The cell repopulation source may comprise undifferentiated or differentiated contractile cells, such as skeletal muscle satellite cells, myoblasts, stem or mesenchymal cells. The implanted cells may be autologous muscle cells, allogenic muscle cells or xenogenic muscle cells.
The cell repopulation source may comprise genetic material optionally contained in a delivery vehicle wherein the delivery vehicle may comprise a nucleic acid molecule, such as plasmid DNA, Further, the plasmid DNA may optionally contains at least one gene. The nucleic acid molecule may encode a gene such as a myogenic determination gene. The delivery vehicle may be delivered in liposomes other any other-suitable source.
These regions of repopulated cells provide improved diastolic cardiac function. Significantly, augmenting the repopulated regions with electrical stimulation provides improved systolic as well as diastolic function. As a result, the present invention provides systems and methods that include a cell repopulation source (i.e., a cell repopulating agent) and an electrical stimulation device (i.e. a stimulation source). The cell repopulation source can include undifferentiated contractile cells such as autologous muscle cells, or nucleic acid for conversion of fibroblasts, for example, to myoblasts. The repopulation source can included differentiated cardiac or skeletal cells, such as cardiomyocytes, myotubes and muscle fiber cells, and the like The cell repopulation source can be delivered by direct injection into the myocardium or via the coronary vasculature. Cell repopulation can be carried out using a syringe, or alternatively, a delivery device such as a catheter can be used. The cells or genetic material can be delivered simultaneously with the electrical stimulation device, or they can be delivered separately. Preferably, the electrical stimulation device is the carrier of the cells or genetic material. The electrical stimulation device typically includes an implantable muscle stimulator and electrodes. Significantly, it does not include leads connecting it to any other device.
There are a wide variety of methods that can be used to deliver nucleic acid to nonundifferentiated or differentiated contractile cells. For instance such as fibroblast cells, can be convert their phenotype from connective to contractile. Such methods are well known to one of skill in the art of genetic engineering. For example, the desired nucleic acid can be inserted into an appropriate delivery vehicle, such as, for example, an expression plasmid, cosmid, YAC vector, and the like, to produce a recombinant nucleic acid molecule. There are a number of viruses, live or inactive, including recombinant viruses, that can also be used. A retrovirus can be genetically modified to deliver any of a variety of genes. Adenovirus can also be used to deliver nucleic acid capable of converting nonundifferentiated contractile cells to undifferentiated contractile cells, preferably, muscle cells. A “recombinant nucleic acid molecule,” as used herein, is comprised of an isolated nucleotide sequence inserted into a delivery vehicle. Regulatory elements, such as the promoter and polyadenylation signal, are operably linked to the nucleotide sequence as desired.
Almost any delivery vehicle can be used for introducing nucleic acids into the cardiovascular system, including, for example, recombinant vectors, such as one based on adenovirus serotype 5, Ad5, as set forth in French, et al., Circulation, 90, 2414–2424 (1994). An additional protocol for adenovirus-mediated gene transfer to cardiac cells is set forth in WO 94/11506, Johns, J. Clin. Invest., 96, 1152–1158 (1995), and in Barr, et al., Gene Ther., 1, 51–58 (1994). Other recombinant vectors include, for example, plasmid DNA vectors, such as one derived from pGEM3 or pBR322, as set forth in Acsadi, et al., The New Biol., 3, 71–81, (1991), and Gal, et al., Lab. Invest., 68, 18–25 (1993), cDNA-containing liposomes, artificial viruses, nanoparticles, and the like.
Promoters and polyadenylation signals used are preferably functional within the cells of the patient. In order to maximize protein production, regulatory sequences may be selected which are well suited for gene expression in the cardiac cells into which the recombinant nucleic acid molecule is administered. For example, the promoter is preferably a cardiac tissue-specific promoter-enhancer, such as, for example, cardiac isoform troponin C (cTNC) promoter. Parmacek, et al., J. Biol. Chem., 265, 15970–15976 (1990), and Parmacek, et al., Mol. Cell Biol., 12, 1967–1976 (1992). In addition, codons may be selected which are most efficiently transcribed in the cell. One having ordinary skill in the art can produce recombinant nucleic acid molecules which are functional in the cardiac cells.
The cells and/or genetic material can be associated with the carrier as a coating or a preformed film, for example. If desired, the carrier can be initially coated with an adhesive, such as that available under the trade name CELLTAK BIOCOAT Cell Environments available from Stratech Scientific Ltd., Luton, Bedfordshire, United Kingdom, to enhance adhesion of the polymeric matrix containing the undifferentiated and or differentiated contractile cells and/or genetic material
The genetic material and/or undifferentiated and or different tiated contractile cells can also be delivered in a pharmaceutical composition using a catheter, for example. Such pharmaceutical compositions can include, for example, the nucleic acid, in the desired form, and/or cells in a volume of phosphate-buffered saline with 5% sucrose. In other embodiments of the invention, the nucleic acid molecule and/or cells are delivered with suitable pharmaceutical carriers, such as those described in the most recent edition of Remington 's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.
The electrical stimulation device can provide burst stimulation, which is typically used for stimulating skeletal muscle cells, or it can provide synchronous single pulse stimulation, which is typically used for stimulating cardiac muscle cells. Alternatively, the electrical stimulation device can provide both burst and synchronous single pulse stimulation. This is particularly desirable if the new contractile tissue formed includes both skeletal and cardiac muscle cells and/or skeletal muscle cells are initially formed and then converted to cardiac muscle cells. A pressure lead, or other means of monitoring a physiological condition such as wall acceleration or intraventricular pressure, can be used to determine when to switch from burst mode to single phase mode of stimulation. If desired, two electrical stimulation devices can be used, one that provides burst stimulation and one that. provides synchronous single pulse stimulation.
The stimulator 22 can be in the shape of a cylinder, or other appropriate shape suitable for implantation, and of a size sufficiently small for implantation. For example, it can be about 5 mm in diameter and 20 mm in length. Preferred materials include titanium, but other biocompatible materials can also be used. Stimulator 22 may contain a battery or other power source, electronics to detect heart beats and produce burst stimulation, and telemetry circuits for triggering stimulation on demand. Such circuitry can be developed by one of skill in the art, particularly in view of the teachings of U.S. Pat. No. 5,697,884 (Francischelli et al.), U.S. Pat. No. 5,658,237 (Francischelli), U.S. Pat. No. 5,207,218 (Carpentier et al.), U.S. Pat. No. 5,205,810 (Guiraudon et al.), U.S. Pat. No. 5,069,680 (Grandjean), and U.S. Pat. No. 4,411,268 (Cox).
FIG. 3 is a block diagram illustrating various components of a stimulator 22 which is programmable by means of an external programming unit (not shown). One such programmer adaptable for the purposes of the present invention is the commercially available Medtronic Model 9790 programmer. The programmer is a microprocessor device which provides a series of encoded signals to stimulator 22 by means of a programming head which transmits radio frequency encoded signals to IPG 51 according to a telemetry system, such as that described in U.S. Pat. No. 5,312,453 (Wybomy et al.), for example.
Stimulator 22, illustratively shown in FIG. 3, is electrically coupled to the patient's heart 56 by lead 54. Lead 54, which includes two conductors, is coupled to a node 62 in the circuitry of stimulator 22 through input capacitor 60. In the presently disclosed embodiment, an activity sensor 63 provides a sensor output to a processing/amplifying activity circuit 65 of input/output circuit 68. Input/output circuit 68 also contains circuits for interfacing with heart 56, antenna 66, and circuit 74 for application of stimulating pulses to heart 56 to moderate its rate under control of software-implemented algorithms in microcomputer unit 78.
Antenna 66 is connected to input/output circuit 68 to permit uplink/downlink telemetry through RF transmitter and receiver unit 55. Unit 55 may correspond to the telemetry and program logic disclosed in U.S. Pat. No. 4,556,063 (Thompson et al.), or to that disclosed in the above-referenced Wybomy et al. patent. Voltage reference (VREF) and bias circuit 61 generates a stable voltage reference and bias current for the analog circuits of input/output circuit 68. Analog-to-digital converter (ADC) and multiplexer unit 58 digitizes analog signals and voltages to provide “real-time” telemetry intracardiac signals and battery end-of-life (EOL) replacement functions.
The electrical stimulation device can include a variety of mechanisms for holding it in place in the myocardium. For example, it can include extendable hooks or talons. Alternatively, the tissue contacting portion of the device can be treated to achieve a microsurface texture (as disclosed by Andreas F. von Recumin in: Biomaterials, 12, 385–389, “Texturing of Polymer Surfaces at the Cellular Level” (1991); Biomaterials, 13, 1059–1069, “Macrophage Response to Microtextured Silicone” (1992); and Journal of Biomedical Materials Research, 27, 1553–1557, “Fibroblast Anchorage to Microtextured Surfaces” (1993)). In an alternative embodiment, the stimulator can be in the form of a screw that is driven into the muscle wall by turning.
Adenovirus expressing myogenin (Myogen adenovirus/cDNA, which can be produced according to the method described by Murry et al., J. Clin. Invest., 98, 2209–2217 (1196)) was injected directly to the myocardium using a 100 microliter syringe. 109 pfu (pfu-plaque forming units-one pfu is approximately 50 adenovirus particles) were diluted with saline to form a 100 microliter solution. This solution was kept on dry ice until the injection, and delivered in four equal amounts to the perimeter of the infarct zone, 90 degrees apart.
81.6% M199 (Sigma, M4530)
7.4% MEM (Sigma, M-4655)
10% Fetal Bovine Serum (Hyclone, Cat.#A-1115-L)
B. Split ratios of 1:4–1:6 will yield a confluent monolayer within 96 hours.
ml of Trypsin Solution
M. To maintain a healthy culture, change medium every 2–3 days.
B. Count cells using a hemocytometer. The most accurate range for the hemocytometer is between 20–50 cells/square.
A. Isolation Medium: 80.6% M199 (Sigma, M-4530), 7.4% MEM (Sigma, M-4655), 10% Fetal Bovine Serum (Hyclone, Cat.#A-1115-L), 2� (2%) Penicillin/Streptomycin (Final Conc. 200,000 U/L Pen./20 mg/L Strep., Sigma, P-0781).
B. Myoblast Growth Medium: 81.6% M199 (Sigma, M-4530), 7.4% MEM (Sigma, M-4655), 10% Fetal Bovine Serum (Hyclone, Cat.#A-1115-L), 1� (1%) Penicillin/Streptomycin (Final Conc. 100,000 U/L Pen./10 mg/L Strep., Sigma, P-0781).
Add approximately 30 ml to a 50 ml sterile centrifuge tube (10 gm biopsy or less). Add approximately 50 ml to a 125 ml sterile media bottle (up to 25 gm biopsy). B. Place the Isolation Medium on ice or ice packs to keep cold (approximately 4� C.). C. Prepare the enzyme solution, the same day it will be used, by adding 1.0 gm collagenase and 0.2 gm hyaluronidase to 100 ml of M199 (100 ml of enzyme/disbursing solution is enough to digest 40–50 gm of skeletal muscle). D. Filter sterilize the enzyme solution first through a 0.45 μm filter and then a 0.22 μm filter and keep at 4� C. until ready to use. E. Prepare the disbursing solution, the same day it will be used, by adding 1 gm of the protease to 100 ml of M199. F. Filter sterilize through a 0.22 μm filter and keep at 4� C. until ready to use. G. Under semi-sterile conditions remove the skeletal muscle biopsy, preferably from the belly of the muscle, and place it into the isolation medium. H. Seal the container and store at approximately 4� C. until ready to mince. I. Remove the tissue and place into a sterile petri dish. J. Trim off any connective tissue and measure the final weight. K. Rinse the tissue with sterile 70% EtOH for 30 seconds. L. Aspirate the EtOH and rinse the tissue 2� with HBSS. M. Finely mince the biopsy using scissors and tweezers. N. Transfer the minced biopsy into 50 ml sterile centrifuge tubes. No more than 20 gm/tube to allow for effective enzymatic digestion. O. Rinse the tissue by adding approximately 25 ml/tube of HBSS, mix, and pellet the tissue by centrifuging at 2000 RPM (allow the centrifuge to reach 2000 RPM and turn off). P. Decant off the HBSS and repeat the rinse and centrifuge an additional two more times. Q. Add enzyme solution to the tubes (approximately 25 ml/15 gm—20 gm original biopsy). R. Incubate tubes in the incubator shaker for 20 minutes (Set Point—37� C., 300 RPM). S. Centrifuge at 2000 RPM for 5 minutes and discard the supernatant. T. Add disbursing solution to the tubes (approximately 25 ml/15 gm—20 gm original biopsy). U. Incubate tubes in the incubator shaker for 15 minutes (Set Point—37� C., 300 RPM). V. Centrifuge at 2000 RPM for 5 minutes. W. Harvest the supernatant, inactivate the enzyme by adding FBS to a final concentration of 10%, and store at 4� C. X. Add disbursing solution to the tubes for a second enzymatic digestion (approximately 25 ml/15 gm—20 gm original biopsy). Y. Incubate tubes in the incubator shaker for 15 minutes (Set Point—37� C., 300 RPM). Z. Centrifuge at 2000 RPM for 5 minutes. AA. Harvest the supernatant and inactivate the enzyme by adding FBS to a final concentration of 10%. BB. Centrifuge the cell slurry from the disbursing digestion steps (refer to W and AA) at 2400 RPM for 10 minutes. CC. Remove and discard the supernatant. DD. Resuspend the cell pellets in a minimal volume of Wash Solution. EE. Combine the pellets in a 50 ml centrifuge tube, bring the volume up to 40 ml using Wash Solution. FF. Centrifuge at 2400 RPM for 10 minutes. GG. Remove the supernatant and repeat the cell wash two more times. HH. On the final rinse resuspend the pellet in 2 ml of MEM. If the initial biopsy was close to or greater than 25 gm resuspend into 4 ml of MEM. II. Prepare 20% Percoll and 60% Percoll in MEM. JJ. Make the density gradient by layering 10 ml of 20% Percoll/MEM over 5 ml of 60% Percoll/MEM (refer to FIG. 1). KK. Add 2 ml of the cell suspension on the top of the 20% Percoll band. LL. Use a scale to prepare a second tube as a counter balance for centrifugation. MM. Centrifuge at 11947 RPM (15000�g) for 5 minutes at 8� C. (adjust acceleration to 5 and brake to 0). NN. Isolate the band of cells that develops between the 20% and 60% Percoll layers. This band contains the myoblast cells. OO. Determine the volume of the band and dilute it with 5 volumes of growthmedium. Note: If the Percoll isn't diluted with enough growth medium it will be very difficult to pellet the myoblasts out of solution.
PP. Centrifuige at 3000 RPM for 10 minutes. QQ. Remove the supernatant and resuspend the pellet in growth medium. RR. Count the cells in suspension. SS. Plate out the cells in the BIOCOAT Laminin coated T-flasks at approximately 1�104 cells/cm2. The first plating should be done on a laminin coated surface to aid in cell attachment. TT. Culture the cells to 60%–80% confluence. If the cells are allowed to become confluent they will terminally differentiate into myotubes. UU. Trypsinization Procedure:
Wash the monolayer with HBSS Add trypsin (0.5 g/l trypsin) T-Flask
Split ratio's of 1:4 to 1:6 work well for a 60–80% confluent culture.
In order to assure that the transplanted skeletal cells were present at the end of the two week period, preserved tissue sections were analysed with immuno-histochemistry using an anti-myosin antibody (skeletal, fast, MY-32). Positive (green) staining at two different regions of the ablated site indicated the presence of the injected skeletal muscle cells in the ablated region of myocardium, two weeks after their introduction. This immuno-staining study provided definitive evidence for the presence of skeletal muscle cells in the myocardium. The immuno-histochemistry staining protocol used is described as follows:
Polyclonal Rabbit Anti-Connexin-43, Zymed, Cat.No. 71-0700. Goat Anti-Mouse IgG-FITC, Sigma, Cat.No. F-O257. Goat Anti-Rabbit IgG (Whole Molecule)-TRITC, Sigma, Cat.No. T-6778. PBS, Sigma, Cat.No. 1000-3. Goat Serum, Sigma. Acetone, Sigma, Cat.No. A-4206. Mounting Medium, Sigma Cat.No. 1000-4. Microscope, Nikon, Labophot-2. Samples:
Skeletal Muscle (Control) Posterior Lesion Mid Lesion Anterior Lesion J(L) Ventricular Free Wall (Control)
A. Clean glass slides with 95% EtOH and treat with poly-Lysine or buy pre-treated slides. B. Obtain tissue samples and freeze onto cryostat chucks. C. Cut 8 μm thick cryostat sections of the frozen tissue block, place on treated glass slides, and store at ≦−70� C. D. Allow tissue sections to come to room temperature prior to initiating staining (approximately 15–30 minutes). E. Fix samples in cold Acetone (≦−10� C.) for 10 minutes at 4� C. F. Wash sample with PBS three times (care must be taken to avoid washing the sample off of the slide). G. Block samples with 10% Goat Serum/PBS for 20 minutes at room temperature, using a humidified chamber. H. Dilute the first primary antibody, Connexin-43, 1:100 in PBS containing 10% goat serum. Dilute enough antibody to cover the samples (approximately 150 μl), add to the tissue sections, and incubate in a humidified chamber for 1 hour at room temperature. I. Wash sample in 10% Goat Serum/PBS three times (5 minutes/wash). J. Dilute the second primary antibody, My-32, 1:200 in PBS containing 10% goat serum. Dilute enough antibody to cover the samples (approximately 150 μl), add to the tissue sections, and incubate in a humidified chamber for 1 hour at room temperature K. Wash samples in 10% Goat Serum/PBS three times (5 minutes/wash). L. Dilute the secondary antibodies, mix the antibody solutions, and add to the tissue sections.
Anti-Rabbit IgG (Whole Molecule)-TRITC, 1:50 in PBS. Anti-Mouse IgG-FITC, 1:100 in PBS. M. Incubate in a dark, humidified chamber, for 45 minutes at room temperature. N. Wash samples in PBS three times (5 minutes/wash). O. Add mounting medium and a coverslip. P. Read on the microscope using the FITC filter, the TRITC filter, and the UV light source. Q. Store samples in a dark chamber at ≦4� C. The complete disclosures of the patents, patent applications, and publications listed herein are incorporated by reference, as if each were individually incorporated by reference. The above examples and disclosure are intended to be illustrative and not exhaustive. These examples and description will suggest many variations and alternatives to one of ordinary skill in this art. All these alternatives and variations are intended to be included within the scope of the attached claims. Those familiar with the art may recognize other equivalents to the specific embodiments described herein which equivalents are also intended to be encompassed by the claims attached hereto.
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