| 1 | <p align="JUSTIFY"><font face="Times New Roman" size="2"><strong>Part 1: Items that need to be prepared before proceeding to cell plating:</strong></font> |
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| 2 | <dl> |
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| 3 | <dd> |
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| 4 | <p align="JUSTIFY"> <br> |
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| 5 | <dd><font face="Times New Roman" size="2"><strong>1- </strong></font><font face="Times New Roman" size="2">Appropriate volume of complete NSC medium is prepared by mixing </font><font face="Times New Roman" size="2">NeuroCult</font><sup><font face="Times New Roman" size="2">®</font></sup><font face="Times New Roman" size="2"> NSC Basal Medium and NeuroCult</font><sup><font face="Times New Roman" size="2">®</font></sup><font face="Times New Roman" size="2"> NSC Proliferation Supplements at a 9:1 ratio, respectively.</font> |
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| 6 | <dd> <br> |
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| 7 | <dd><font face="Times New Roman" size="2"><strong>2- </strong></font><font face="Times New Roman" size="2">An</font><font face="Times New Roman" size="2"><strong> </strong></font><font face="Times New Roman" size="2">aliquot</font><font face="Times New Roman" size="2"> of </font> |
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| 8 | <!-- Space after “or” Space after “potential” Space “An” “aliquot” “of” --> |
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| 9 | <font face="Times New Roman" size="2">NeuroCult</font><sup><font face="Times New Roman" size="2">®</font></sup><font face="Times New Roman" size="2">NCFC Serum-Free Medium without Cytokines is thawed.</font> |
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| 10 | <dd><br> |
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| 11 | <dd><font face="Times New Roman" size="2"><strong>3- </strong></font><font face="Times New Roman" size="2">The medium is warmed up in a 37ºC water bath.</font> |
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| 12 | <dd> <br> |
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| 13 | <dd><font face="Times New Roman" size="2"><strong>4- </strong></font><font face="Times New Roman" size="2">Stock solutions of epidermal growth factor (EGF), basic fibroblastic growth factor (b-FGF) at the concentration of 10 µg/ml and heparin at 0.2% are prepared ahead. </font> |
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| 14 | <dd><br> |
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| 15 | <dd><font face="Times New Roman" size="2"><strong>5-</strong></font><font face="Times New Roman" size="2"> Depending on the experiment size, several 35mm tissue culture dishes are needed to plate the cells and one 150-200cm plastic Petri dish is also needed to hold the duplicate 35mm dishes and a third 35mm dish for water.</font> |
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| 16 | <dd> <br> |
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| 17 | </dl> |
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| 18 | <p align="JUSTIFY"><font face="Times New Roman" size="2"><strong>Part2: Cell preparation:</strong></font> |
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| 19 | <p align="JUSTIFY"> <br> |
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| 20 | <p align="JUSTIFY"><font face="Times New Roman" size="2">Depending on your experiment, cells can be prepared from an adult or embryonic source (primary dissociated tissue or dissociated neurospheres). Dissect tissues from adult/embryonic mouse central nervous system (CNS) or dissociate adult/embryonic-derived neurospheres as described before </font><sup><font face="Times New Roman" size="2">1,2</font></sup><font face="Times New Roman" size="2"> and then:</font> |
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| 21 | <p align="JUSTIFY"> <br> |
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| 22 | <dl> |
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| 23 | <dd> |
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| 24 | <p align="JUSTIFY"><font face="Times New Roman" size="2"><strong>1- </strong></font><font face="Times New Roman" size="2">T</font><font face="Times New Roman" size="2">he single cell suspension is passed through a 40-µm-size mesh filter so as to remove non-dissociated clumps. </font> |
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| 25 | <dd> |
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| 26 | <p align="JUSTIFY"><br> |
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| 27 | <dd> |
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| 28 | <p align="JUSTIFY"><font face="Times New Roman" size="2"><strong>2- </strong></font><font face="Times New Roman" size="2">10µl of the cell suspension is mixed with 90µl of Trypan blue to perform a cell count. </font><font face="Times New Roman" size="2"><strong>Note</strong></font><font face="Times New Roman" size="2">: Other appropriate cell dilutions can also be used.</font> |
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| 29 | <dd> |
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| 30 | <p align="JUSTIFY"> <br> |
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| 31 | <dd> |
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| 32 | <p align="JUSTIFY"><font face="Times New Roman" size="2"><strong>3- </strong></font><font face="Times New Roman" size="2">If using</font><font face="Times New Roman" size="2"><strong> </strong></font><font face="Times New Roman" size="2">primary embryonic or adult derived neural cells, dilute the cell suspension to a concentration of 6.5</font><font face="Symbol"></font><font face="Times New Roman" size="2">10</font><sup><font face="Times New Roman" size="2">5</font></sup><font face="Times New Roman" size="2"> cells/ml in complete NSC medium. If using cells from dissociated neurospheres derived from adult or embryonic neural cells, dilute the cell suspension to a concentration of 2.2 </font><font face="Symbol"></font><font face="Times New Roman" size="2">10</font><sup><font face="Times New Roman" size="2">5 </font></sup><font face="Times New Roman" size="2">cells/ml in complete NSC medium.</font> |
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| 33 | <dl> |
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| 34 | <dd> <br> |
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| 35 | <dd><br> |
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| 36 | </dl> |
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| 37 | </dl> |
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| 38 | <p align="JUSTIFY"><font face="Times New Roman" size="2"><strong>Part2: Plating cells in semi-solid N-CFCA medium:</strong></font> |
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| 39 | <p align="JUSTIFY"> <br> |
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| 40 | <dl> |
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| 41 | <dl> |
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| 42 | <dd> |
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| 43 | <p align="JUSTIFY"><font face="Times New Roman" size="2"><strong>1- </strong></font><font face="Times New Roman" size="2">The</font><font face="Times New Roman" size="2"><strong> </strong></font><font face="Times New Roman" size="2">appropriate volume of the following components is mixed in order, depending on the number of replicates. Here we mix the amount needed for two replicates or duplicate dishes. For additional numbers of replicates, please refer to </font><font face="Times New Roman" size="2"><strong>table 1</strong></font><font face="Times New Roman" size="2">.</font> |
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| 44 | </dl> |
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| 45 | </dl> |
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| 46 | <ul> |
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| 47 | <li> |
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| 48 | <p align="JUSTIFY"> <font color="#231f20" face="Times New Roman"><font size="2">1.7 ml of NeuroCult</font></font><font color="#231f20" face="Times New Roman"><font size="2">® </font></font><font color="#231f20" face="Times New Roman"><font size="2">NCFC Serum-Free Medium without Cytokines</font></font> |
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| 49 | <li> |
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| 50 | <p align="JUSTIFY"> <font color="#231f20" face="Times New Roman"><font size="2">330 </font></font><font face="Times New Roman" size="2">µl of </font><font color="#231f20" face="Times New Roman"><font size="2">NeuroCult</font></font><font color="#231f20" face="Times New Roman"><font size="2">® </font></font><font color="#231f20" face="Times New Roman"><font size="2">NSC Proliferation Supplements </font></font> |
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| 51 | <li> |
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| 52 | <p align="JUSTIFY"><font color="#231f20" face="Times New Roman"><font size="2">6.6 </font></font><font face="Times New Roman" size="2">µl of EGF (10µg/ml)</font> |
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| 53 | <li> |
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| 54 | <p align="JUSTIFY"> <font face="Times New Roman" size="2">32 </font><font face="Times New Roman" size="2">µl of </font><font face="Times New Roman" size="2">Penicillin/Streptomycin</font> |
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| 55 | <li> |
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| 56 | <p align="JUSTIFY"> <font face="Times New Roman" size="2">3.3 </font><font face="Times New Roman" size="2">µl of b-FGF (10µg/ml) required </font><font face="Times New Roman" size="2"><strong>only</strong></font><font face="Times New Roman" size="2"> </font><font face="Times New Roman" size="2"><strong>if culturing cells derived from adult neural cells</strong></font><font face="Times New Roman" size="2">.</font> |
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| 57 | <li> |
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| 58 | <p align="JUSTIFY"> <font face="Times New Roman" size="2">3.3 </font><font face="Times New Roman" size="2">µl of heparin (0.2%) required </font><font face="Times New Roman" size="2"><strong>only</strong></font><font face="Times New Roman" size="2"> </font><font face="Times New Roman" size="2"><strong>if culturing cells derived from adult neural cells.</strong></font> |
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| 59 | <li> |
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| 60 | <p align="JUSTIFY"> <font face="Times New Roman" size="2">25</font><font face="Times New Roman" size="2"> µl of cell suspension (of 2.2 </font><font face="Symbol"></font><font face="Times New Roman" size="2"> 10</font><sup><font face="Times New Roman" size="2">5</font></sup><font face="Times New Roman" size="2"> cells/ml cells from dissociated neurospheres or 6.5 </font><font face="Symbol"></font><font face="Times New Roman" size="2">10</font><sup><font face="Times New Roman" size="2">5</font></sup><font face="Times New Roman" size="2"> cells/ml primary cells from dissociated CNS tissue). </font><font face="Times New Roman" size="2"><strong>Note</strong></font><font face="Times New Roman" size="2">: The final cell numbers plated should be about 2500 cells per 35 mm culture dish for neurosphere derived cells and 7500 cells per 35 mm culture dish for primary cells. In some cases, the final cell plating density has to be adjusted by performing a cell titration experiment. For statistical analyses, the total number of colonies detected after 21 days of culture should be within the range of at least 50 – 150 colonies.</font> |
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| 61 | </ul> |
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| 62 | <dl> |
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| 63 | <dl> |
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| 64 | <dl> |
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| 65 | <dd> |
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| 66 | <p align="JUSTIFY"><br> |
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| 67 | </dl> |
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| 68 | <dd> |
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| 69 | <p align="JUSTIFY"><font face="Times New Roman" size="2"><strong>2- </strong></font><font face="Times New Roman" size="2">T</font><font face="Times New Roman" size="2">he medium containing the cells is mixed gently and then 1.3 ml of cold Collagen solution is transferred to the cell suspension and mixed well by gentle pipetting to avoid introducing any bubbles. </font> |
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| 70 | <dd> |
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| 71 | <p align="JUSTIFY"><br> |
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| 72 | <dd> |
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| 73 | <p align="JUSTIFY"><font face="Times New Roman" size="2"><strong>3- </strong></font><font face="Times New Roman" size="2">T</font><font face="Times New Roman" size="2">he mixed solution is dispensed to the center of each 35mm culture dish (~1.5 ml/dish) and the dishes are tipped gently using a circular motion to let the mixture spread evenly over the surface of the dish. .</font> |
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| 74 | <dd> |
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| 75 | <p align="JUSTIFY"> <br> |
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| 76 | <dd> |
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| 77 | <p align="JUSTIFY"><font face="Times New Roman" size="2"><strong>4- </strong></font><font face="Times New Roman" size="2">The duplicate</font><font face="Times New Roman" size="2"><strong> </strong></font><font face="Times New Roman" size="2">35 mm culture dishes are placed into a 100 mm Petri plate. The lid of new 35 mm dish is removed and the open dish is also placed in the same 100 mm Petri dish. Sterile water is added to this open 35 mm dish to maintain optimal humidity during the incubation period. Square bioassay plates (245 mm) are used when more replicate 35 mm dishes have been set up. Again, include 2 or 3 open 35 mm dishes containing sterile water.</font> |
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| 78 | </dl> |
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| 79 | </dl> |
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| 80 | <p align="JUSTIFY"> <br> |
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| 81 | <dl> |
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| 82 | <dl> |
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| 83 | <dd><font face="Times New Roman" size="2"><strong>5- </strong></font><font face="Times New Roman" size="2">The plates are transferred to an incubator set at 37°C, 5% CO</font><font face="Times New Roman" size="2">2 </font><font face="Times New Roman" size="2">and 95% humidity. </font><font face="Times New Roman" size="2">The collagen congeals by increasing temperature and gel formation will occur within approximately one hour. The cultures should not be disturbed during this time.</font> |
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| 84 | <dd> |
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| 85 | <p align="JUSTIFY"> <br> |
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| 86 | <dd> |
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| 87 | <p align="JUSTIFY"><font face="Times New Roman" size="2"><strong>6- </strong></font><font face="Times New Roman" size="2">Cultured cells are incubated for 21 days (differences in colony size can be clearly distinguished after 21 days).</font> |
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| 88 | <dd> |
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| 89 | <p align="JUSTIFY"> <br> |
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| 90 | </dl> |
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| 91 | </dl> |
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| 92 | <p align="JUSTIFY"><font face="Times New Roman" size="2"><strong>Part4: Preparing replenishment medium and feeding the culture:</strong></font> |
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| 93 | <dl> |
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| 94 | <dl> |
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| 95 | <dl> |
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| 96 | <dd> |
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| 97 | <p align="JUSTIFY"> <br> |
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| 98 | </dl> |
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| 99 | <dd> |
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| 100 | <p align="JUSTIFY"><font face="Times New Roman" size="2">As N-CFCA cultures are incubated for an extended period of time (21 days), cultures should be fed with the appropriate complete NeuroCult</font><sup><font face="Times New Roman" size="1">® </font></sup><font face="Times New Roman" size="2">replenishment medium prepared fresh as follows:</font> |
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| 101 | </dl> |
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| 102 | </dl> |
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| 103 | <p align="JUSTIFY"> <br> |
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| 104 | <ul> |
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| 105 | <li> |
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| 106 | <p align="JUSTIFY"><font face="Times New Roman" size="2">4.5ml of NeuroCult</font><sup><font face="Times New Roman" size="2">®</font></sup><font face="Times New Roman" size="2"> </font><font face="Times New Roman" size="2">NSC Basal Medium is mixed with 0.5ml of NeuroCult</font><font face="Times New Roman" size="2">® </font><font face="Times New Roman" size="2">NSC Proliferation Supplements and then the growth factors is added:</font> |
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| 107 | <li> |
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| 108 | <p align="JUSTIFY"> <font face="Times New Roman" size="2">250 μL of 10μg/ml EGF (to give a final concentration of 0.5μg/ml EGF)</font> |
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| 109 | <li> |
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| 110 | <p align="JUSTIFY"> <font face="Times New Roman" size="2">125 μL of 10μg/mL b-FGF (to give a final concentration of 0.25μg/ml b-FGF) </font><font face="Times New Roman" size="2"><strong>required only</strong></font><font face="Times New Roman" size="2"> </font><font face="Times New Roman" size="2"><strong>if culturing cells derived from adult neural cells</strong></font><font face="Times New Roman" size="2">.</font> |
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| 111 | <li> |
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| 112 | <p align="JUSTIFY"> <font face="Times New Roman" size="2">125 μL of 0.2% Heparin, </font><font face="Times New Roman" size="2"><strong>required only</strong></font><font face="Times New Roman" size="2"> </font><font face="Times New Roman" size="2"><strong>if culturing cells derived from adult neural cells</strong></font><font face="Times New Roman" size="2">.</font> |
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| 113 | <li> |
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| 114 | <p align="JUSTIFY"> <font face="Times New Roman" size="2">60 μL of the appropriate complete NeuroCult</font><font face="Times New Roman" size="1">® </font><font face="Times New Roman" size="2">Replenishment Medium (for embryonic or adult cells) is added into the center of each NCFC Assay dish once every 7 days during the entire NCFC Assay culture incubation (21 days).</font> |
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| 115 | </ul> |
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| 116 | <dl> |
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| 117 | <dl> |
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| 118 | <dl> |
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| 119 | <dl> |
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| 120 | <dl> |
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| 121 | <dd> |
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| 122 | <p align="JUSTIFY"><br> |
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| 123 | </dl> |
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| 124 | </dl> |
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| 125 | </dl> |
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| 126 | </dl> |
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| 127 | </dl> |
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| 128 | <p align="JUSTIFY"><font face="Times New Roman" size="2"><strong>Part5: Scoring N-CFC Assay derived colonies and calculating the frequency of </strong></font><font face="Times New Roman" size="2"><em><strong>bona fide</strong></em></font><font face="Times New Roman" size="2"><strong> NSCs and neural progenitor cells:</strong></font> |
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| 129 | <dl> |
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| 130 | <dl> |
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| 131 | <dd> |
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| 132 | <p align="JUSTIFY"> <br> |
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| 133 | </dl> |
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| 134 | </dl> |
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| 135 | <ul> |
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| 136 | <li> |
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| 137 | <p align="JUSTIFY"><font face="Times New Roman" size="2">Each 35 mm culture dish is placed on a gridded scoring dish with the grid size of 2.0 mm </font><font face="Symbol" size="2"></font><font face="Times New Roman" size="2"> 2.0 mm and then both dishes are placed on the microscope stage. </font> |
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| 138 | <li> |
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| 139 | <p align="JUSTIFY"><font face="Times New Roman" size="2">Using low power (2.5X - 5X) objective lens, each dish is scanned and the colonies are scored based on their sizes.</font> |
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| 140 | <li> |
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| 141 | <p align="JUSTIFY"> <font face="Times New Roman" size="2">Colonies are classified into two major categories: </font> |
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| 142 | </ul> |
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| 143 | <ol> |
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| 144 | <li> |
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| 145 | <p align="JUSTIFY"><font face="Times New Roman" size="2">Less than 2 mm diameter </font> |
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| 146 | <li> |
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| 147 | <p align="JUSTIFY">≥<font face="Times New Roman" size="2">2 mm diameter</font> |
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| 148 | </ol> |
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| 149 | <dl> |
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| 150 | <dl> |
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| 151 | <dd><br> |
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| 152 | <dd><br> |
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| 153 | </dl> |
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| 154 | </dl> |
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| 155 | <p align="JUSTIFY"><font face="Times New Roman"><strong>Representative Results:</strong></font> |
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| 156 | <p align="JUSTIFY"> <br> |
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| 157 | <p align="JUSTIFY"><font face="Times New Roman" size="2">As in neurosphere assay, cells plated in N-CFC assay start to proliferate and make small colonies of cells within 3 - 7 days after plating (Figure 1). In two weeks time, colonies of different sizes can be distinguished. While the majority of colonies tend to stop growing after 14 days, some colonies continue to increase in size. By day 21, colonies can be classified into one of the four categories: 1) less than 0.5 mm diameter, 2) 0.5 - 1 mm diameter, 3) 1 - 2 mm diameter and 4) ≥2mm in diameter. Practically, colonies smaller than 2 mm diameter are referred to as progenitor derived (Figure 2) and colonies ≥2mm in diameter are referred to as NSC derived (Figure 3). The number of colonies ≥2mm in diameter per total cells plated, represents the frequency of the actual </font><font face="Times New Roman" size="2"><em>bona fide</em></font><font face="Times New Roman" size="2"> neural stem cells with long-term self-renewal and multi-potential capabilities. The total neurosphere forming frequency in a particular cell population after 7-8 day in a parallel NSA experiment has been estimated to be similar to the total colony forming frequency of the same cell population after 21 days in a N-CFCA experiment. The N-CFCA however, provides a more permissive condition so as each cell can show its full proliferative potential.</font> |
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| 158 | <p align="JUSTIFY"> <br> |
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| 159 | <p align="JUSTIFY"><br> |
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| 160 | <p align="JUSTIFY"><font face="Times New Roman" size="2"><strong>Table 1. </strong></font><font face="Times New Roman" size="2">Components of complete N-CFC assay culture.</font> |
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| 161 | <p align="JUSTIFY"> <br> |
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| 162 | <table width="687" border="1" cellpadding="8" cellspacing="0"> |
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| 163 | <col width="306"> |
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| 164 | <col width="96"> |
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| 165 | <col width="103"> |
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| 166 | <col width="116"> |
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| 167 | <tr valign="TOP"> |
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| 168 | <td width="306" height="24"><p align="JUSTIFY"><font face="Times New Roman"><strong>Component</strong></font></td> |
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| 169 | <td width="96"><p align="JUSTIFY"><font face="Times New Roman"><strong>2 replicates</strong></font></td> |
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| 170 | <td width="103"><p align="JUSTIFY"><font face="Times New Roman"><strong>3 replicates</strong></font></td> |
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| 171 | <td width="116"><p align="JUSTIFY"><font face="Times New Roman"><strong>4 replicates</strong></font></td> |
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| 172 | </tr> |
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| 173 | <tr valign="TOP"> |
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| 174 | <td width="306"><p><font color="#231f20" face="Times New Roman"><font size="2">NeuroCult</font></font><font color="#231f20" face="Times New Roman"><font size="2">® </font></font><font color="#231f20" face="Times New Roman"><font size="2">NCFC Serum-Free Medium without Cytokines</font></font></td> |
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| 175 | <td width="96"><p align="JUSTIFY"><font face="Times New Roman" size="2">1700</font></td> |
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| 176 | <td width="103"><p align="JUSTIFY"><font face="Times New Roman" size="2">2550</font></td> |
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| 177 | <td width="116"><p align="JUSTIFY"><font face="Times New Roman" size="2">3400</font></td> |
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| 178 | </tr> |
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| 179 | <tr valign="TOP"> |
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| 180 | <td width="306"><p align="JUSTIFY"><font color="#231f20" face="Times New Roman"><font size="2">NeuroCult</font></font><font color="#231f20" face="Times New Roman"><font size="2">® </font></font><font color="#231f20" face="Times New Roman"><font size="2">NSC Proliferation Supplements</font></font> |
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| 181 | <p align="JUSTIFY"> <br> |
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| 182 | </td> |
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| 183 | <td width="96"><p align="JUSTIFY"><font face="Times New Roman" size="2">330</font></td> |
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| 184 | <td width="103"><p align="JUSTIFY"><font face="Times New Roman" size="2">495</font></td> |
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| 185 | <td width="116"><p align="JUSTIFY"><font face="Times New Roman" size="2">660</font></td> |
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| 186 | </tr> |
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| 187 | <tr valign="TOP"> |
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| 188 | <td width="306"><p align="JUSTIFY"><font color="#231f20" face="Times New Roman"><font size="2">EGF (10 μg/mL)</font></font></td> |
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| 189 | <td width="96"><p align="JUSTIFY"><font face="Times New Roman" size="2">6.6</font> |
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| 190 | <p align="JUSTIFY"> <br> |
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| 191 | </td> |
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| 192 | <td width="103"><p align="JUSTIFY"><font face="Times New Roman" size="2">9.9</font></td> |
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| 193 | <td width="116"><p align="JUSTIFY"><font face="Times New Roman" size="2">13.2</font></td> |
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| 194 | </tr> |
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| 195 | <tr valign="TOP"> |
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| 196 | <td width="306"><p align="JUSTIFY"><font color="#231f20" face="Times New Roman"><font size="2">bFGF (10 μg/mL), </font></font><font color="#231f20" face="Times New Roman"><font size="2"><strong>only for adult cells</strong></font></font></td> |
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| 197 | <td width="96"><p align="JUSTIFY"><font face="Times New Roman" size="2">3.3</font> |
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| 198 | <p align="JUSTIFY"> <br> |
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| 199 | </td> |
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| 200 | <td width="103"><p align="JUSTIFY"><font face="Times New Roman" size="2">4.95</font></td> |
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| 201 | <td width="116"><p align="JUSTIFY"><font face="Times New Roman" size="2">6.6</font></td> |
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| 202 | </tr> |
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| 203 | <tr valign="TOP"> |
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| 204 | <td width="306"><p><font color="#231f20" face="Times New Roman"><font size="2">Heparin Solution (0.2%), </font></font><font color="#231f20" face="Times New Roman"><font size="2"><strong>only for adult cells</strong></font></font></td> |
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| 205 | <td width="96"><p align="JUSTIFY"><font face="Times New Roman" size="2">3.3</font> |
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| 206 | <p align="JUSTIFY"> <br> |
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| 207 | </td> |
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| 208 | <td width="103"><p align="JUSTIFY"><font face="Times New Roman" size="2">4.95</font></td> |
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| 209 | <td width="116"><p align="JUSTIFY"><font face="Times New Roman" size="2">6.6</font></td> |
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| 210 | </tr> |
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| 211 | <tr valign="TOP"> |
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| 212 | <td width="306"><p align="JUSTIFY"><font face="Times New Roman" size="2">Penicillin/Streptomycin (1:100)</font></td> |
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| 213 | <td width="96"><p align="JUSTIFY"><font face="Times New Roman" size="2">32</font></td> |
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| 214 | <td width="103"><p align="JUSTIFY"><font face="Times New Roman" size="2">48</font></td> |
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| 215 | <td width="116"><p align="JUSTIFY"><font face="Times New Roman" size="2">64</font></td> |
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| 216 | </tr> |
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| 217 | <tr valign="TOP"> |
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| 218 | <td width="306"><dl> |
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| 219 | <dt><font color="#231f20" face="Times New Roman"><font size="2">Cells at:</font></font> |
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| 220 | <dt> <font color="#231f20" face="Times New Roman"><font size="2">2.2 x 10</font></font><font color="#231f20" face="Times New Roman"><sup><font size="2">5</font></sup></font><font color="#231f20" face="Times New Roman"><font size="2"> </font></font><font color="#231f20" face="Times New Roman"><font size="2">cultured cells/mL OR</font></font> |
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| 221 | </dl> |
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| 222 | <p align="JUSTIFY"> <font color="#231f20" face="Times New Roman"><font size="2">6.5 x 10</font></font><font color="#231f20" face="Times New Roman"><sup><font size="2">5</font></sup></font><font color="#231f20" face="Times New Roman"><font size="2"> </font></font><font color="#231f20" face="Times New Roman"><font size="2">primary cells/mL</font></font></td> |
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| 223 | <td width="96"><p align="JUSTIFY"><font face="Times New Roman" size="2">25</font></td> |
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| 224 | <td width="103"><p align="JUSTIFY"><font face="Times New Roman" size="2">37.5</font></td> |
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| 225 | <td width="116"><p align="JUSTIFY"><font face="Times New Roman" size="2">50</font></td> |
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| 226 | </tr> |
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| 227 | <tr valign="TOP"> |
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| 228 | <td width="306"><p align="JUSTIFY"><font color="#231f20" face="Times New Roman"><font size="2">Collagen Solution</font></font></td> |
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| 229 | <td width="96"><p align="JUSTIFY"><font face="Times New Roman" size="2">1300</font></td> |
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| 230 | <td width="103"><p align="JUSTIFY"><font face="Times New Roman" size="2">1950</font></td> |
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| 231 | <td width="116"><p align="JUSTIFY"><font face="Times New Roman" size="2">2600</font></td> |
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| 232 | </tr> |
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| 233 | </table> |
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