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HomePrimary Cell CultureProtocol for Preparation and Cultivation of PromoCell® Cryopreserved and Proliferating Cells

Protocol for Preparation and Cultivation of PromoCell® Cryopreserved and Proliferating Cells

These products are available in the US, Canada, and select European countries.

Protocol for Handling and Cultivating Cryopreserved Primary Cells

Important Notes:

  • Upon arrival, store the cryopreserved cells in liquid nitrogen, or seed them immediately. Storage at -80 °C is not sufficient for cell preservation and causes irreversible cell damage.
  • Handle using aseptic technique and a laminar flow biosafety cabinet.
  1. Prepare the medium
    Calculate the area of culture surface needed according to the plating density (see table below) and the lot-specific cell numbers stated on the certificate of analysis. Fill the appropriate volume of PromoCell® Growth Medium (at least 9 mL per vial of cells) in cell culture vessels. Place the vessels in an incubator (37 °C, 5% CO2) for 30 minutes.

  2. Thaw the cells
    Remove the cryovial from the liquid nitrogen container and immediately place on dry ice - even for short transportation. Under a laminar flow cabinet, briefly twist the cap a quarter turn to relieve pressure, then re-tighten. Immerse the vial into a water bath (37 °C) just up to the screw cap for 2 minutes. Ensure that no water enters the thread of the screw cap.

  3. Disinfect the vial and seed the cells
    Thoroughly rinse the cryovial with 70 % ethanol in a laminar flow cabinet. Then, aspirate the excess ethanol from the thread area of the screw cap. Open the vial and transfer the cells to a cell culture vessel containing the prewarmed medium from step 1.

  4. Incubate the cells
    Place the vessel in an incubator (37 °C, 5% CO2) for cell attachment. Replace the medium after 16–24 hours and every two to three days thereafter. The cells should be sub-cultured according to the subcultivation protocol (see below), once they have reached 70 – 90 % confluency.

Protocol for Handling and Cultivating Proliferating Primary Cells

PromoCell primary cells are available in a proliferating (non-cryopreserved) format.

Important notes:

  • Start immediately after delivery.
  • Use aseptic technique and work in a laminar flow biosafety cabinet.
  1. Incubate the cells
    Unpack the culture vessel, do not open the cap, and immediately place it in an incubator (37 °C, 5% CO2) for 3 hours to allow the cells to recover from transportation.

  2. Replace the transport medium
    Carefully open the vessel, rinse the inner side of the lid with 70% ethanol, and let air dry. Aspirate the transport medium from the vessel. Add 10 mL of the appropriate PromoCell® Cell Growth Medium.

  3. Check and incubate the cells
    Check the cell density. Open the cap half a turn and place the vessel in an incubator (37 °C, 5% CO2). Change the medium every two to three days. The cells should be subcultured according to the subcultivation protocol (see below), once they have reached 70 - 90% confluency.

Primary Cell Subcultivation Protocol

Important Notes:

  • Use aseptic technique and a laminar flow biosafety cabinet.
  • Due to their low-serum content or the absence of serum, PromoCell® media are not suitable for trypsin neutralization (when subculturing the cells, for example). Instead, we recommend using the DetachKit (C-41200, C-41210, C-41220), which contains HEPES BSS, Trypsin/EDTA and Trypsin Neutralizing Solution.
  1. Prepare the reagents and wash the cells
    Place the PromoCell® DetachKit at room temperature for at least 30 minutes to adjust the temperature of the reagents. Carefully aspirate the medium from the culture vessel. Add 100 µL HEPES BSS Solution per cm2 of vessel surface to wash the cells and agitate the vessel carefully for 15 seconds.

  2. Detach the cells
    Note: We recommend detaching the cells at room temperature.

    Carefully aspirate the HEPES BSS from the culture vessel. Add 100 µL Trypsin/EDTA Solution per cm2 of vessel surface. Close the vessel and examine the cells under a microscope. When the cells begin to detach, gently tap the side of the culture vessel to dislodge the remaining cells.

  3. Neutralize the trypsin and harvest the cells
    Add 100 µL Trypsin Neutralization Solution per cm2 of vessel surface and gently rock the vessel to mix. Carefully aspirate the cell suspension by pipette and transfer to a centrifugation tube. Spin down the cells for three minutes at 220 x g.

  4. Incubate the cells
    Discard the supernatant from the cell pellet. Add 1 mL of the appropriate PromoCell® Cell Growth Medium and resuspend the cells by gently pipetting up and down. Plate the cells according to the recommended seeding density in new cell culture vessels containing prewarmed Cell Growth Medium. Place the vessels in an incubator (37 °C, 5% CO2) and change the media every two to three days.
Product #*DescriptionRecommended Culture Media*Plating DensityPassage after ThawingMarkerPopulation Doublings
C-12810Human Cardiac Myocytes (HCM)C-2207010,000 - 15,000 cells per cm²P2Sarcomeric alpha-Actinin+ Slow muscle myosin+> 15
C-12980Human Pericytes from Placenta (hPC-PL)C-280403,000 – 4,000 cells per cm²P2
C-12620Human Nasal Epithelial Cells (HNEpC)C-2106010,000 - 15,000 cells per cm²P2Cytokeratin+> 15
C-12644Human Tracheal Epithelial Cells (HTEpC)C-2106010,000 - 15,000 cells per cm²P2Cytokeratin+> 15
C-12640Human Bronchial Epithelial Cells (HBEpC)C-2106010,000 - 15,000 cells per cm²P2Cytokeratin+> 15
C-12642Human Small Airway Epithelial Cells (HSAEpC)C-2107010,000 - 15,000 cells per cm²P2Cytokeratin+> 15
C-12665Human Renal Epithelial Cells (HREpC)C-26001 C-2603010,000 - 15,000 cells per cm²P2Cytokeratin+> 15
C-12660Human Renal Cortical Epithelial Cells (HRCEpC)C-26001 C-2603010,000 - 15,000 cells per cm²P2Cytokeratin+> 15
C-12001Normal Human Epidermal Keratinocytes (NHEK), juvenile foreskin, single donorC-200115,000 cells per cm²P2Cytokeratin+> 15
C-12005Normal Human Epidermal Keratinocytes (NHEK), juvenile foreskin, pooledC-200115,000 cells per cm²P2Cytokeratin+> 15
C-12003Normal Human Epidermal Keratinocytes (NHEK), adult, single donorC-200115,000 cells per cm²P2Cytokeratin+> 15
C-12006Normal Human Epidermal Keratinocytes (NHEK), adult, pooledC-200115,000 cells per cm²P2Cytokeratin+> 15
C-12710Human Chondrocytes (HCH)C-2710110,000 - 20,000 cells per cm²P2Differentiation tested> 10
C-12533Human Aortic Smooth Muscle Cells (HAoSMC)C-220627,500 - 10,000 cells per cm²P2Smooth muscle a-actin+> 15
C-12511Human Coronary Artery Smooth Muscle Cells (HCASMC)C-220627,500 - 10,000 cells per cm²P2Smooth muscle a-actin+> 15
C-12521Human Pulmonary Artery Smooth Muscle Cells (HPASMC)C-220627,500 - 10,000 cells per cm²P2Smooth muscle a-actin+> 15
C-12500Human Umbilical Artery Smooth Muscle Cells (HUASMC)C-220627,500 - 10,000 cells per cm²P2Smooth muscle a-actin+> 15
C-12565Human Tracheal Smooth Muscle Cells (HTSMC)C-220627,500 - 10,000 cells per cm²P2Smooth muscle a-actin+> 15
C-12561Human Bronchial Smooth Muscle Cells (HBSMC)C-220627,500 - 10,000 cells per cm²P2Smooth muscle a-actin+> 15
C-12575Human Uterine Smooth Muscle Cells (HUtSMC)C-220627,500 - 10,000 cells per cm²P2Smooth muscle a-actin+> 15
C-12530Human Skeletal Muscle Cells (SkMC)C-23060 C-230613,500 - 7,000 cells per cm²P2Sarcomeric myosin+ Differentiation tested> 15
C-12300Normal Human Dermal Fibroblasts (NHDF), juvenile foreskinC-230103,500 - 7,000 cells per cm²P2CD90+> 15
C-12302Normal Human Dermal Fibroblasts (NHDF), adult donorC-230203,500 - 7,000 cells per cm²P2CD90+> 15
C-12360Human Pulmonary Fibroblasts (HPF)C-230203,500 - 7,000 cells per cm²P2CD90+> 15
C-12380Human Aortic Adventitial Fibroblasts (HAoAF)C-230203,500 - 7,000 cells per cm²P2CD90+> 10
C-12375Human Cardiac Fibroblasts (HCF)C-230253,500 - 7,000 cells per cm²P2CD90+ Smooth muscle α-actin- Slow muscle myosin-> 15
C-12385Human Uterine Fibroblasts (HUF)C-230203,500 - 7,000 cells per cm²P2CD90+> 15
C-12720Human Osteoblasts (HOB)C-27001 C-2702010,000 – 20,000 cells per cm²P2Alkaline phosphatase+ Mineralization tested> 10
C-12200Human Umbilical Vein Endothelial Cells (HUVEC), single donorC-22010 C-220115,000 –10,000 cells per cm²P1vW+ CD31+ Dil-Ac-LDL uptake+> 15
C-12203Human Umbilical Vein Endothelial Cells (HUVEC), pooledC-22010 C-220115,000 –10,000 cells per cm²P1vWF+ CD31+ Dil-Ac-LDL uptake+> 15
C-12206Human Umbilical Vein Endothelial Cells (HUVEC) isolated in Growth Medium 2, single donorC-220115,000 –10,000 cells per cm²P1vWF+ CD31+ Dil-Ac-LDL uptake+> 15
C-12208Human Umbilical Vein Endothelial Cells (HUVEC) isolated in Growth Medium 2, pooledC-220115,000 –10,000 cells per cm²P1vWF+ CD31+ Dil-Ac-LDL uptake+> 15
C-12205Human Umbilical Vein Endothelial Cells (HUVEC), pre-screenedC-22010 C-220115,000 –10,000 cells per cm²P1vWF+ CD31+ Dil-Ac-LDL uptake+ VEGF response> 15
C-12202Human Umbilical Artery Endothelial Cells (HUAEC)C-22010 C-220115,000 –10,000 cells per cm²P1vWF+ CD31+ Dil-Ac-LDL uptake+> 15
C-12271Human Aortic Endothelial Cells (HAoEC)C-22020 C-220225,000 –10,000 cells per cm²P2vWF+ CD31+ Dil-Ac-LDL uptake+> 15
C-12221Human Coronary Artery Endothelial Cells (HCAEC)C-22020 C-220225,000 –10,000 cells per cm²P3vWF+ CD31+ Dil-Ac-LDL uptake+> 15
C-12241Human Pulmonary Artery Endothelial Cells (HPAEC)C-22010 C-220115,000 –10,000 cells per cm²P2vWF+ CD31+ Dil-Ac-LDL uptake+> 15
C-12231Human Saphenous Vein Endothelial Cells (HSaVEC)C-22010 C-220115,000 –10,000 cells per cm²P2vWF+ CD31+ Dil-Ac-LDL uptake+> 15
C-12210Human Dermal Microvascular Endothelial Cells (HDMEC), juvenile foreskinC-22020 C-2202210,000 - 20,000 cells per cm²P2vWF+ CD31+ Dil-Ac-LDL uptake+> 15
C-12212Human Dermal Microvascular Endothelial Cells (HDMEC), adult donorC-22020 C-2202210,000 - 20,000 cells per cm²P2vWF+ CD31+ Dil-Ac-LDL uptake+> 15
C-12215Human Dermal Microvascular Endothelial Cells (HDMEC), pre-screenedC-22020 C-2202210,000 - 20,000 cells per cm²P2vWF+ CD31+ Dil-Ac-LDL uptake+ VEGF response+> 15
C-12211Human Dermal Blood Endothelial Cells (HDBEC), juvenile foreskinC-2202010,000 - 20,000 cells per cm²P2Podoplanin- CD31+> 15
C-12225Human Dermal Blood Endothelial Cells (HDBEC), adult donorC-2202010,000 - 20,000 cells per cm²P2Podoplanin- CD31+> 15
C-12216Human Dermal Lymphatic Endothelial Cells (HDLEC), juvenile foreskinC-2202210,000 - 20,000 cells per cm²P2Podoplanin+ CD31+> 15
C-12217Human Dermal Lymphatic Endothelial Cells (HDLEC), adult donorC-2202210,000 - 20,000 cells per cm²P2Podoplanin+ CD31+> 15
C-12285Human Cardiac Microvascular Endothelial Cells (HCMEC)C-22020 C-2202210,000 - 20,000 cells per cm²P2vWF+ CD31+ Dil-Ac-LDL uptake+> 10
C-12281Human Pulmonary Microvascular Endothelial Cells (HPMEC)C-22020 C-2202210,000 - 20,000 cells per cm²P2vWF+ CD31+ Dil-Ac-LDL uptake+> 15
C-12295Human Uterine Microvascular Endothelial Cells (HUtMEC)C-22020 C-2202210,000 - 20,000 cells per cm²P2vWF+ CD31+ Dil-Ac-LDL uptake+> 15
C-12400Normal Human Epidermal Melanocytes (NHEM), juvenile foreskinC-240105,000 - 10,000 cells per cm²P2Mel-5+> 15
C-12402Normal Human Epidermal Melanocytes (NHEM), juvenile foreskin, cultured in M2 MediumC-243005,000 - 10,000 cells per cm²P2Mel-5+> 15
C-12403Normal Human Epidermal Melanocytes (NHEM), adult donor, cultured in M2 MediumC-243005,000 - 10,000 cells per cm²P2Mel-5+> 15
C-12730Human White Preadipocytes (HWP) subcutaneousC-27410 C-27436 C-274385,000 cells per cm²P2Differentiation tested> 10
C-12732Human White Preadipocytes (HWP) visceralC-27410 C-27436 C-274385,000 cells per cm²P2Differentiation tested> 10

*catalog numbers in this table are for cryopreserved cells and media in ready-to-use packaging

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