PRIMARY CELL CULTURE GUIDETHAWING, FREEZING AND SUBCULTIVATION

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Freezing

Ensure that your culture is in good condi-tion

In order to preserve cell vitality after thaw-ing, it is recommended to use cells in a low passage (P3-4) that haven’t undergone many population doublings.

Check cell confluence

Cells should be in the logarithmic growing phase when processed for cryopreservation. Therefore, cells should be harvested ideally at approximately 80% confluence. Freezing cells at a higher confluence will lead to loss of vitality and poor attachment rate after thawing.

Determine cell number

In order to obtain a single cell suspension follow the steps 1 - 9 of the subcultivation protocol (see on the right).Subsequently, discard the supernatant (1. red arrow), add 1 ml of the PromoCell Cryo-SFM cryopreservation medium at room temperature (2. red arrow), and resus-pend the cells by carefully pipetting up and down. Count the cells and dilute at mini-mum 1 x 106 up to 4 x 106 cells per ml for the subsequent freezing process (lower cell numbers are feasible, if using a con-trolled-rate freezer). Transfer the diluted cells into appropriate cryovials using the recommended volume.

Freezing process

A slow and continuous cooling rate of -1°C/min is optimal for achieving the best possible recovery after cryopreservation. For this, a controlled-rate freezer will lead to best results. Alternatively, a conventional isopropanol freezing container (e.g. Mr. Frosty™) can also be used to achieve an approximate cooling rate of -1°C/min.

If using an isopropanol freezing container:• Fill the recommended volume of isopropa-nol as stated in the outer part of the contain-er at room temperature.• Use only suitable cryogenic vials which fit tightly into the vial inserts to ensure good heat dissipation.• Insert your cryovials and store the freezing container at -80°C overnight.• Transfer cryopreserved cells from -80°C directly to liquid nitrogen the next day.

Freezing Protocol

Critical points:

• Cell confluence at ~ 80%

• Ideal cooling rate: -1°C per minute

• > 1 x 106 cell/ml when freezing manually

• Long-term storage at -80°C is not sufficient for cell preservation

Subcultivation

Prepare the reagents and wash the cells

Place the PromoCell DetachKit at room tem-perature for at least 30 minutes to adjust the temperature of the reagents. Carefully aspi-rate 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.

Detach the cells

Carefully aspirate the Hepes BSS from the culture vessel. Add 100 µl Trypsin/EDTA Solution per cm2 of vessel surface. Note: We recommend detaching the cells at room temperature. Close the vessel and examine the cells under a microscope. When the cells start to detach, gently tap the side of the vessel to loosen the remaining cells.

Neutralize the trypsin and harvest the cells

Add 100 µl Trypsin Neutralization Solution per cm2 of vessel surface and gently agitate. Carefully aspirate the cell suspension and transfer it to a centrifugation tube. Spin down the cells for 3 minutes at 220 x g.

Incubate the cells

Discard the supernatant (step 10.1), add 1 ml of the appropriate PromoCell Cell Growth Medium (step 10.2), and resuspend the cells by carefully pipetting up and down. Plate the cells according to the recommend-ed seeding density (see Tab. 1) in new cell culture vessels containing prewarmed Pro-moCell Cell Growth Medium. Place the vessels in an incubator (37°C, 5% CO2) and change the media every two to three days.

TNS

Hepes BSS Trypsin TNS

TNS

Trypsin EDTA

Hepes BSS

Hepes BSS

Subcultivation Protocol

Critical points:

• Subcultivation between 70 - 90% confluence

• Trypsin quality, concentration and temperature

• Incubation time

• Trypsin inhibition

Thawing

Prepare the medium

Calculate the needed culture surface area according to the plating density (see Tab. 1) 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.

Thaw the cells

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

Disinfect the vial and seed the cells

Thoroughly rinse the cryovial with 70% ethanol under a laminar flow bench. 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 con-taining the prewarmed medium from step 1.

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 subcultured, according to the subcultivation protocol (see below), once they have reached 70 - 90% confluency.

37°C

Protocol for Cryopreserved Cells

10,000 – 20,000 cells per cm2

5,000 cells per ml1 x 106 cells per ml1 x 106 cells per ml

5,000 – 10,000 cells per cm2

Plating densityCell type

Endothelial cells (large vessels)HUVEC, HUAEC, HAoEC, HCAEC, HPAEC, HSaVEC

3,500 – 7,000 cells per cm2FibroblastsNHDF, HPF, HAoAF, HCF, HUF

5,000 cells per cm2KeratinocytesNHEK

10,000 – 20,000 cells per cm2OsteoblastsHOB

3,500 – 7,000 cells per cm2Skeletal muscle cellsSkMC

4,000 cells per cm2Human mesenchymal stem cellshMSC

Endothelial cells (microvascular)HDMEC, HDBEC, HDLEC, HCMEC, HPMEC, HUtMEC

10,000 – 15,000 cells per cm2Epithelial cellsHNEpC, HTEpC, HBEpC, HSAEpC, HREpC, HRCEpC, HPIEpC

10,000 – 15,000 cells per cm2Cardiac myocytesHCM

5,000 – 10,000 cells per cm2Follicle dermal papilla cellsHFDPC

5,000 – 10,000 cells per cm2MelanocytesNHEM

5,000 cells per cm2PreadipocytesHWP

7,500 – 10,000 cells per cm2Smooth muscle cellsHAoSMC, HCASMC, HPASMC, HUASMC, HTSMC, HBSMC, HUtSMC

4,000 cells per cm2Human pericyteshPC-PL

10,000 – 20,000 cells per cm2ChondrocytesHCH

Human stem and blood cellshCD34+-CBhMoCD14+-PBhMNC

Table 1: Recommmended plating densities of the human primary cells and the human stem and blood cells available at PromoCell are listed below.

Critical points:

• Prewarming medium

• Transport on dry ice

• Cryovial disinfection

• Water bath maintenance

• DMSO toxicity