Cor.At. Cardiomyocytes derived from Mouse Embryonic Stem Cells. Technical Manual

Transcription

1 Cor.At Cardiomyocytes derived from Mouse Embryonic Stem Cells Technical Manual Axiogenesis AG Nattermannallee 1, D Köln Tel.: , Fax:

2 Table of contents 1.0 Introduction Cor.At Cardiomyocytes for Pharmacological and Toxicological Screening Cor.At Cardiomyocytes Compared to Cell Lines and Primary Cells Materials and Reagents Cor.At Product Specification Additional Materials and Reagents Required for Cor.At Cardiomyocyte Culture Safety Instructions Thawing & Plating Cor.At Cardiomyocytes Vials Media Preparation Washing & Counting Cells Plating Cells on Fibronectin-Coated Dishes Well Plates Media Preparation Culture of Cor.At Cardiomyocytes in 96-well Plates Maintenance of Cor.At cells for Long Term Culture (Day 2 to Day 14) Characteristics of Cor.At Cardiomyocytes in Culture Figures Figure 1. Layout of Cor.At cells on the fibronectin-coated 96-well plate Figure 2. Process Flowchart Helpful Hints Fibronectin-coated dishes Representative Pictures of Cor.At Cardiomyocyte Cultures at 6, 24, 48 Hours, Day 3 and Day Figure 3. Green fluorescent Cor.At cardiomyocytes; 6 hours of culture (100X) Figure 4. Cor.At cardiomyocytes; 24 hours of culture: A) GFP+ ; B) phase contrast (100X) Figure 5. Green fluorescent Cor.At cardiomyocytes: A) 48 hours of culture; B) day 3 of culture Figure 6. Green fluorescent Cor.At Cardiomyocytes: A) day 9 of culture; B) day 14 of culture References Limited Use Label License info@axiogenesis.com, Page 2 of 16

3 1.0 Introduction Cor.At cardiomyocytes are derived from transgenic mouse embryonic stem cells. These cells are puromycin resistant and have the green fluorescent protein (GFP) reporter gene driven by a cardiacspecific promoter (αmhc promoter). 1 Cor.At cells are produced through in vitro differentiation of mouse embryonic stem (ES) cells and puromycin selection of cardiomyocytes. Through BrdU incorporation assays, Cor.At cells were determined to have limited proliferative capacity similar to primary cells. These highly pure cells (>99% purity) express cardiac-specific connexin-43, an indication of the ability for electric coupling of these cells, as shown in immunostaining. Patch clamp analyses, as well as multi-electrode array (MEA) recordings, demonstrate the normal electrophysiological properties of these cells. Further transplantation of these ES cell-derived cardiomyocytes into infarcted heart resulted in teratoma-free engraftment of the GFP+ cells Cor.At Cardiomyocytes for Pharmacological and Toxicological Screening Cor.At cardiomyocytes are particularly useful for cell-based in vitro assays in pharmacology and toxicology. These cells are ideal for high content screening, as well as, for medium to high throughput screening. Cor.At cells can be used for: Safety pharmacology, toxicological analysis of compounds (e.g. herg assay) Screening for pharmacological effects Drug development Electrophysiology, patch clamp and MEAs Molecular and cell biology Tissue Engineering and transplantation Research & development 1.2 Cor.At Cardiomyocytes Compared to Cell Lines and Primary Cells Cor.At cardiomyocytes have many advantages over primary cells and cell lines. These cells are highly standardised and are 99.9% pure. The culture and maintenance of Cor.At cardiomyocytes require minimal laboratory time when compared to the culture and maintenance of rat neonatal cells. 4 Reproducible results can be expected for every assay. These homogeneous cells are: Standardised from lot-to-lot 99.9% pure and have fully functional cardiac phenotype Absolutely no fibroblast contamination Express all relevant cardiac ion channels (K+, Ca2+, Na+) and shows predictive response to cardiotoxic compounds when compared to clinical data info@axiogenesis.com, Page 3 of 16

4 Cells can be purchased frozen in different formulations in cryo vials: Catalog numbers Ax-C-MC01 (ES derived GFP positive), Ax-C-MC02 (ES derived colorless), Ax-C-MC03 (ips derived colorless) or preplated in fibronectin-coated 96-well plates: Catalog number Ax-C-MC01-96w. Cells are cultured for 48 hours to allow them to recover post thaw and should be cultured in 35 mm or 60 mm BD BioCoat Fibronectin-coated dishes (Catalog # for 35 mm dishes or Catalog # for 60 mm dishes). For coating plates and dishes of alternate sizes, we recommend coating the dish with Sigma Fibronectin from bovine plasma (1mg/mL solution), Catalog #F Materials and Reagents Please read the entire technical manual before beginning the culture of mouse cardiomyocytes. Additional information on stability and storage instructions for the cells can be found in the specific Product Information Sheets (PIS). 2.1 Cor.At Product Specification Catalog # Product Description Unit / Format Storage Conditions 1 million viable Cor.At cardiomyocytes > 99.9% pure Cryo vial Liquid nitrogen * (6 months) -80 C * (4 weeks) Ax-C-MC01-1M Cor.At Cells (Vials) Culture medium for Cor.At cardiomyocytes, 100 ml 100 ml bottle -20 C * (6 months) 4 C * (2 weeks) Puromycin stock solution, 50uL (10 mg/ml) 1mL vial -20 C * (6 months) 4 C (1 week) 96-well plates pre-plated with Cor.At cardiomyocytes 20,000 cells per well > 99.9% pure Fibronectin-coated 96- well plate -80 C * (4 weeks) Plates should not be stored below 80 C! Ax-C-MC01-96w Cor.At Cells (Plates) Culture medium for Cor.At cardiomyocytes, 250 ml 250 ml bottle -20 C * (6 months) 4 C * (2 weeks) Thawing medium for Cor.At cardiomyocytes, 30 ml 60 ml bottle -20 C * (6 months) 4 C * (2 weeks) Ax-M-MC250E (Europe, ROW) Ax-M-MC250N (USA and Japan) Cor.At Culture Medium * Refer to PIS for additional information. Puromycin stock solution, 50 ul (10 mg/ml) Culture medium for Cor.At cardiomyocytes, 250 ml 1 ml vial 250 ml bottle -20 C * (6 months) 4 C (1 week) -20 C * (6 months) 4 C * (2 weeks) info@axiogenesis.com, Page 4 of 16

5 2.2 Additional Materials and Reagents Required for Cor.At Cardiomyocyte Culture Vertical laminar flow hood certified for Level I handling of biological materials Incubator with humidity and gas control to maintain 37 C and 95% humidity in an atmosphere of 5% CO 2 in air 37 C water bath Inverted fluorescence microscope with Green Fluorescent Protein (GFP) or Fluorescein Isothiocyanate (FITC) filters (FITC filter recommended for viewing cultures but not mandatory) Sterile 50 ml polypropylene (PP) tubes Centrifuge with rotor for 50 ml PP tubes (not required for 96-well plate format) 8-channel or 12-channel micropipette (e.g. 8-channel Eppendorf "Research Pro" 1200) for 96- well plate format Sterile pipette tips BD BioCoat Fibronectin-coated dishes (Catalog # for 35 mm dishes or Catalog # for 60 mm dishes; not required 96-well plate format) Sigma Fibronectin from bovine plasma (1mg/mL solution), Catalog #F-1141 (for coating alternate sized dishes and plates) Trypan blue solution (e.g. Sigma-Aldrich, Catalog #T8154) PBS without Ca ++ and Mg ++ Neubauer hemacytometer 3.0 Safety Instructions The kit is intended for in vitro Research Use Only The kit is not intended for Diagnostic, Therapeutic or Clinical Use and is not approved for human in vivo applications. Cor.At cells are genetically modified mouse cells and should be handled according to local directives (Biosafety level 1). Cor.At cells can be inactivated by autoclaving at 121 C for 20 minutes. Cor.At cardiomyocytes should be cultured in a sterile environment according to good cell culture and good laboratory practices. It is highly recommended that gloves and lab coats be worn when handling all reagents as some reagents contain chemicals that may be harmful. Please consult the PIS and Material Safety Data Sheets (MSDS) for additional safety instructions where applicable. info@axiogenesis.com, Page 5 of 16

6 4.0 Thawing & Plating Cor.At Cardiomyocytes 4.1 Vials Media Preparation 1. Thaw Cor.At cutlure medium (Catalog #06099) and the puromycin stock solution at 4 C overnight or at room temperature (RT; C). It is recommended to thaw the culture medium in the dark. Do not attempt to thaw the Cor.At culture medium at 37 C because precipitation of the proteins in the medium may occur. The culture medium contains light sensitive compounds and should be kept in the dark while thawing. 2. Pipette 50 ml of the thawed Cor.At culture medium into a sterile 50 ml PP tube and add 50 µl of puromycin stock solution. Mix well and warm the medium to 37 C The puromycin is used as a selecting agent. The Cor.At culture medium containing puromycin is required for medium change at 24 hours of culture. 3. Pipette another 8 ml of Cor.At culture medium into a second sterile 50 ml PP tube. Do not add puromycin to this tube. This medium is used to wash the cells once the cells are thawed. 4. Store the remaining Cor.At culture medium at 4 C. The remaining medium is used for medium changes after 48 hours (or 2 days) of culture and can be kept at 4 C for up to three weeks. Do not use Cor.At culture medium if it has been opened and stored at 4 C for more than three weeks Washing & Counting Cells 1. Add 50 µl Trypan Blue solution to 40 µl PBS (without Ca++ and Mg++) in a small tube (e.g. Eppendorf tube). 2. Prepare the hemacytometer by cleaning the chambers and coverslip with isopropanol. Wipe the hemacytometer dry using lint-free tissue. 3. Remove the Cor.At vial(s) out of the dry ice box or the 80 C freezer. Immediately place the vial(s) in a 37 C water bath for two minutes. Remove the vial(s) from the water bath and wipe with 70% isopropanol or ethanol to sterilize. 4. Immediately pipette the cell suspension into the second 50 ml tube containing 8 ml of Cor.At medium without puromycin (prepared in Step 3, Section 4.1.1) to wash the cells. info@axiogenesis.com, Page 6 of 16

7 5. Use 1 ml of Cor.At culture medium to rinse the vial. This ensures that any cells remaining in the vial are also recovered. Add the 1 ml of cells to the 50 ml tube (there should now be ~9 ml of cell suspension in the tube). 6. Centrifuge cells at 200 x g for 5 minutes at room temperature (15 25 C). 7. Aspirate the supernatant, leaving the cell pellet intact and resuspend the cells in 500 ul of Cor.At culture medium containing Puromycin (prepared in Step 2, Section 4.1.1) in preparation for cell counts. 8. Pipette 10 µl of cells into the Trypan Blue solution (prepared in Step 1), mix well, and incubate for 5 minutes at 37 C (i.e. place tube into the incubator). This is a 1 in 10 dilution of the cells. 9. After 5 minutes, fill the Neubauer hemacytometer with the cell suspension, making sure that the chambers do not get under-filled or over-filled (use 10 ul of cells). Count both the clear, refractile cells and the blue colored cells in the 4 large outer quadrants. This is the total number of cells (N) in the four large, outer quadrants. 10. Subsequently, count the number of blue cells (D) in the same four, large outer quadrants; these are the non-viable cells that have incorporated the trypan blue dye. Because of the characteristic decrease in cell membrane integrity in non-viable cells, the trypan blue dye is able to pass through the cell membrane. 11. Calculate the average number of total cells (A N ) by dividing N by 4 (N/4) and the average number of non-viable cells (A D ) by dividing D by 4 (D/4). A N = N 4 and A D = D Calculate the absolute number of viable cells (V) using the formula: V = 10 x (A N A D ) x 10 4 x 0.5 ml AVERAGE CELL COUNT PER SQUARE x DILUTION FACTOR x 10 4 = CELL COUNT PER ml Neubauer hemacytometer showing dimensions of each square. info@axiogenesis.com, Page 7 of 16

8 4.1.3 Plating Cells on Fibronectin-Coated Dishes BD BioCoat Fibronectin-coated dishes (Catalog # for 35 mm dishes or Catalog # for 60 mm dishes) are recommended for the culture of Cor.At cardiomyocytes. Prior to plating the cells onto the BD BioCoat fibronectin-coated dishes, hydrate the dish by aliquoting the appropriate amount (for the exact volume of medium required to coat the dish, refer to the table below) of the Cor.At culture medium (Catalog #06099) into the dish and place the dish into the 37 C incubator for 1 hour. For coating your own 35 mm, 60 mm, 96-well or other multi-well dishes and plates, we recommend using Sigma Fibronectin from bovine plasma (1mg/mL solution), Catalog #F Perform a 1:100 dilution of the fibronectin in PBS without Ca ++ and Mg ++. Please see Section 8.1: Helpful Hints for the coating protocol (page 13). 1. Adjust the cell concentration to ~1 x 10 5 viable cells per cm 2 of the fibronectin-coated dish using Cor.At medium containing Puromycin (prepared in Step 1, Section 4.1.1) as outlined in the table below: Dish / Multiwell Plate Surface area (cm 2 ) Volume of Fibronectin solution for coating (µl) Cor.At cells per well (x 10 6 )* Volume of Medium (ml) 35 mm dish mm dish , well of a 6 well plate well of a 12 well plate well of a 24 well plate well of a 48 well plate well of a 96 well plate Plate the cells on the fibronectin-coated culture dishes, making sure that cells uniformly cover the bottom surface area of the dish to form a monolayer of cells. 3. Place the dish into the 37 C incubator and culture cells at 37 C, 5% CO 2 and 95% humidity for 24 hours. 4. After 24 hours, check the dish under the microscope to ensure that cells have attached to the bottom surface of the dish as illustrated in Figure 4 (Section 8.2). Perform a medium change by removing the existing medium and adding fresh Cor.At culture medium containing puromycin (prepared in Step 2, Section 4.1.1). 5. Return the dish into the 37 C incubator (5% CO 2 and 95% humidity) for another 24 hours. 6. At 48 hours of culture, perform a medium change by removing the existing medium from the dish and adding fresh Cor.At culture medium without puromycin. Cor.At cardiomyocytes must be cultured in puromycin-containing culture medium for at least 48 hours before culture medium without puromycin can be used for medium changes. info@axiogenesis.com, Page 8 of 16

9 7. Cells are now ready to be used but can be cultured for up to 3 weeks with a medium change every other day (i.e. every 48 hours) using Cor.At culture medium without puromycin Well Plates Media Preparation 1. Thaw the Cor.At culture medium (Catalog #06099), the Cor.At thawing medium and the puromycin stock solution at 4 C overnight or at RT (15-25 C). It is recommended to thaw the culture medium and thawing medium in the dark. Do not attempt to thaw the Cor.At medium at 37 C because precipitation of the proteins in the medium may occur. The culture medium and thawing medium contain light sensitive compounds and should be kept in the dark while thawing. 2. Warm the Cor.At culture medium and Cor.At thawing medium at 37 C once the media have thawed. 3. Add 30 µl of the puromycin solution to the 30 ml bottle of pre-warmed Cor.At thawing medium and mix well. Transfer the medium to a sterile tube or reagent reservoir suitable for an 8-channel or 12-channel pipette. 4. Add 50 ml of the Cor.At culture medium into a sterile 50 ml PP tube and add 50 µl of puromycin stock solution. Mix well and warm the medium to 37 C This medium is used for medium changes at 6 hours and 24 hours of culture. 5. Store the remaining Cor.At culture medium at 4 C. The remaining medium is used for medium changes after 48 hours (or 2 days) of culture and can be kept at 4 C for up to three weeks. Do not use Cor.At culture medium if it has been opened and stored at 4 C for more than three weeks Culture of Cor.At Cardiomyocytes in 96-well Plates 1. Remove the Cor.At 96-well plates out of the dry ice box or the -80 C freezer and immediately pipette 225 µl of Cor.At thawing medium containing puromycin (prepare in Step 3, Section 4.2.1) into each well of the 96-well plate. Place the 96-well plate into the 37 C incubator (with 5% CO2, 95% humidity) for 6 hours. The cells in the 96-well plate should still be frozen when the thawing medium is added to the plate. Do not attempt to thaw the cells prior to the addition of the thawing medium. For optimal cell adherence, the plate should not be moved for first 6 hours. 2. Thirty minutes prior to the end of the first 6 hours of culture, warm the 50 ml tube with Cor.At culture medium containing puromycin (prepared in Section 4.2.1, Step 4) to 37 C. Transfer 25 ml of the warmed medium containing puromycin to a sterile tube or reagent reservoir large enough to fit an 8-channel or 12-channel pipette. info@axiogenesis.com, Page 9 of 16

10 3. Remove the Cor.At 96-well plate from the incubator. Gently remove the medium from the 60 inner wells using a slow-running suction pump equipped with an 8-channel or 12-channel pipette tip or a multichannel pipette. It is extremely important that the tips do not touch the bottom of the wells during aspiration. Hold plates at a 45 angle and place pipette tips at the inner rim of the wells. 4. Add 200 µl of warmed medium containing puromycin to each of the 60 inner wells of the plate using a multi-channel pipette such as the 8-channel Eppendorf "Research Pro" 1200 at the slowest speed setting. Ensure that each of the 36 outer wells of the plate have at least 200 µl of liquid in them. If required, add buffer to each of the 36 wells. The liquid is to ensure that the humidity is equally distributed throughout the plate. These outer wells should not have cells in them. 5. Return the plate into the 37 C incubator (with 5% CO 2, 95% humidity) and culture for an additional 18 hours (i.e. a total of 24 hours in culture). 6. At 24 hours of culture, check the dish under the microscope to ensure that cells have attached to the bottom surface of the dish as illustrated in Figure 4 in Section 8.2 then perform a medium change using Cor.At culture medium containing puromycin by repeating steps 2-4 above (Section 4.2.2). 7. Return the plate into the 37 C incubator (with 5% CO 2, 95% humidity) and culture for an additional 24 hours (i.e. total 48 hours). At 48 hours of culture, perform another medium change using Cor. At culture without puromycin and repeat steps 2 4 again. Cor.At cardiomyocytes must be cultured in puromycin-containing culture medium for at least 48 hours before culture medium without puromycin can be used for medium changes. 5.0 Maintenance of Cor.At cells for Long Term Culture (Day 2 to Day 14) After 48 hours the majority of cells should be attached and beating (Figure 5A, Section 8.2). Change the medium using warm Cor.At medium without puromycin every other day after the initial 48 hours in culture. Visually inspect the cells before and after each medium change to ensure that the cells are healthy. The synchronous beating of the cells may temporarily stop after medium changes or if the cells are left at room temperature (15 C - 25 C) for more than 10 minutes. The cells will recover and begin beating once they are returned to 37 C. Cor.At cardiomyocytes can be used after 2 days of culture. For electrophysiology experiments (patch clamp, MEA), we recommend using the cells within the first week of culture. Cells can be cultured up to 21 days post thaw with medium changes every other day. It is not recommended to culture the cells for more than 21 days. Harvesting or passaging the cells is not recommended. This is especially true if the cells have been in culture for more than one week. Gap junctions have formed between cells (as shown by Connexin-43 immunostaining) and forcibly dissociating the cells will damage the cells and cause cell death. info@axiogenesis.com, Page 10 of 16

11 6.0 Characteristics of Cor.At Cardiomyocytes in Culture After 24 hours of culture, most of the cells will have adhered to the bottom surface of the cell culture dish (Figure 4A, Section 8.2). Some of the cells will not be adherent and will appear round and refractile (Figure 4B, Section 8.2). Although the cells are not all adherent, connected groups of adherent cells should already show synchronous beating with frequencies between 180 and 240 beats per minute. After one week of culture, the cells should almost form a confluent monolayer. At this stage, a majority of the cells should be connected through connexin-43 tight junctions and synchronous beating should be visible throughout the culture dish. In DNA characterization studies, approximately 20% of the Cor.At cardiomyocytes show DNA synthesis in the first 10 days of culture. The rate of DNA synthesis decreases to less than 1% after 14 days in culture. Given the fact that cell numbers do not increase during the 14 days in culture, the DNA synthesis mainly results in binucleation of the cardiomyocytes, an indication of the maturation of the cardiac cells. After 14 days in culture, the morphology of the cells change to an elongated phenotype (Figure 6B, Section 8.2). The beating frequency of the cardiomyocytes can increase up to 600 beats per minute, a further indication of the increased maturation of the cells. 3 To clearly visualize the synchronous beating of the cardiomyocytes, we recommend viewing the cells at 200X magnification under the microscope. To visualize the level of confluence of the cultures, view cells under the flourescence microscope equipped with the appropriate filters for GFP. info@axiogenesis.com, Page 11 of 16

12 7.0 Figures 7.1 Figure 1. Layout of Cor.At cells on the fibronectin-coated 96-well plate A blank blank blank blank blank blank blank blank blank blank blank blank B blank blank C blank blank D blank blank E blank 75µl of cell suspension seeded and frozen blank F blank blank G blank H blank blank Add 200 µl of PBS or medium to all "blank" wells blank blank blank 7.2 Figure 2. Process Flowchart info@axiogenesis.com, Page 12 of 16

13 8.0 Helpful Hints 8.1 Fibronectin-coated dishes We highly recommend using BD BioCoat TM Fibronectin coated culture dishes, Catalog # for 35 mm dishes or Catalog # for 60 mm dishes. BD coated dishes must be hydrated for 1 hour in the 37 C incubator using the appropriate volume of medium (please consult the table below). For coating alternate multiwell plates we recommend purchasing Sigmaʼs Fibronectin from bovine plasma (1mg/mL solution), Catalog #F-1141: 1. Dilute the fibronectin 1:100 in PBS without Ca ++ and Mg Aliquot the appropriate volume of fibronectin into the cultureware according to the table below: Dish / Multiwell Plate Surface area (cm 2 ) Volume of Fibronectin solution for coating (µl) Cor.At cells per well (x 10 6 )* Volume of Medium (ml) 35 mm dish (750) mm dish (1,500) well of a 6 well plate well of a 12 well plate well of a 24 well plate well of a 48 well plate well of a 96 well plate Place the freshly coated cultureware into the 37 C incubator for at least 3 hours. If the dishes are not immediately used, the dish can be stored for up to 3 days at 4 C. 4. Remove the remaining fibronectin solution immediately before seeding the Cor.At cells using a slow-running suction pump or a multichannel pipette. Do not allow the surface of the cultureware to become dry after fibronectin coating; therefore, do not remove the fibronectin solution until the cells are ready to be plated. Fibronectin is highly susceptible to shear stress. Avoid vigorous pipetting and do not vortex the tube of fibronectin. For additional handling instructions, consult the Sigma PIS. info@axiogenesis.com, Page 13 of 16

14 8.2 Representative Pictures of Cor.At Cardiomyocyte Cultures at 6, 24, 48 Hours, Day 3 and Day 14. Figure 3. Green fluorescent Cor.At cardiomyocytes; 6 hours of culture (100X) Figure 4. Cor.At cardiomyocytes; 24 hours of culture: A) GFP+ ; B) phase contrast (100X) A) B) info@axiogenesis.com, Page 14 of 16

15 Figure 5. Green fluorescent Cor.At cardiomyocytes: A) 48 hours of culture; B) day 3 of culture A) B) Figure 6. Green fluorescent Cor.At Cardiomyocytes: A) day 9 of culture; B) day 14 of culture A) B) info@axiogenesis.com, Page 15 of 16

16 8.3 References 1. Kolossov E, Lu Z, Drobinskaya I, et al. Identification and characterization of embryonic stem cell-derived pacemaker and atrialcardiomyocytes. FASEB. J. April 2005;19(6): Kolossov E, Bostani T, et al., Engraftment of engineered ES cell-derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium. J Exp Med Oct 2;203(10): Xue B, Pamidimukkala J, Hay M. Sex differences in the development of angiotensin II-induced hypertension in conscious mice. Am J Physiol Heart Circ Physiol May; 288(5):H Epub 2004 Dec Kettenhofen R, Bohlen H. Preclinical assessment of cardiac toxicity. Drug Discov Today Aug; 13(15-16): Epub 2008 Jul Limited Use Label License Cor.At cells, including its derivatives or modifications, are produced in part with technology that is the subject of pending German, U.S. and foreign patent applications and/or patents owned or licensed by Axiogenesis AG (hereinafter "Axiogenesis IP"). The purchase of this product conveys to the buyer the non-exclusive, nontransferable right to use the purchased amount of Cor.At cells and the associated Axiogenesis IP for (i) not-forprofit internal research conducted by the buyer (whether the buyer is an academic or for-profit entity), where "Research" means non-commercial uses or activities which (or the results of which) do not generate revenue, and (ii) certain for-profit activities, including lead discovery, testing and/or research and development of products or potential products that may or may not be, or become, commercially available, quality control of commercial products, and provision of services consisting of these applications to third parties. Axiogenesis AG hereby waives its right to any compensation beyond the price paid by purchaser for Cor.At cells when Cor.At are used for the activities permitted by this license, including the right to any intellectual property or any profits generated by purchaser through purchasers use, directly or indirectly, of Cor.At cells. Accordingly, Axiogenesis AG will not assert a claim against the buyer of infringement of its relevant patents based upon the manufacture, use or sale of a product (including a therapeutic, clinical diagnostic, vaccine, prophylactic or research product) developed in research by the buyer in which Cor.At cells were employed, provided that neither Cor.At cells nor any of its components were used directly in the manufacture of such product. This limited use license does NOT include the right to resell the product, use Cor.At cells directly in the manufacture of a product or as a component of a product, reverse engineer Cor.At cells or Axiogenesis IP, or to use Axiogenesis IP in any way that is separated from intact Cor.At cells (including the isolation and/or use of integrated genetic constructs that represent, in whole or in part, Axiogenesis IP). For information on the foregoing patents or patent applications or on purchasing a license to use Axiogenesis IP for purposes other than those permitted above, contact Business Development Department, Axiogenesis AG, Nattermannalle 1 / S 20, Cologne, Germany. info@axiogenesis.com, Tel: , Fax: info@axiogenesis.com, Page 16 of 16