post-thaw membrane integrity for HUVECs and porcine corneal
endothelial cells [19]. The monolayer cryopreservation protocol
[19] is described in detail below.
Briefly, our optimized protocol for cryopreservation of endothelial monolayers after culture for 7 days on fibronectin-coated
Rinzl coverslips involves loading of CPA (5% DMSO + 6%
HES + 2% chondroitin sulfate) for 15 min on ice, ice nucleation
at À5
C, cooling to À45
C (at a cooling rate of 1
C/min or
0.2
C/min), and plunging into, and storage in, liquid nitrogen.
Upon rapid thawing of cryopreserved HUVEC monolayers in a
37
C water bath and removal of the CPA by serial dilution, we
obtained an absolute viability of 96.6 Æ 2% when the cooling rate
was 1
C/min and 97.3 Æ 3.2% when the cooling rate was 0.2
C/
min [19]. For porcine corneal endothelial cell monolayers, we
obtained an absolute viability of 91.1 Æ 7.6% when the cooling
rate was 1
C/min and 107.8 Æ 7.8% when the cooling rate was
0.2
C/min to a plunge temperature of À40
C [19] (see Note 1).
2 Materials
2.1 Equipment
1. Analytical balance.
2. Biological safety cabinet.
3. Pipette aid and pipettors.
4. Phase contrast microscope (Labovert, Leitz).
5. Coulter counter or hemocytometer.
6. Centrifuge (Eppendorf 5810R tabletop centrifuge, Hamburg,
Germany) for cell passaging.
7. Programmable methanol cooling bath (FTS Systems, Stone
Ridge, NY, USA).
8. T-type thermocouple and OMB-DAQ-55 data acquisition
module (OMEGA Engineering Inc., Stamford, CT, USA).
9. Water bath.
10. Timer.
11. Liquid nitrogen (LN 2 ) dewar or styrofoam box with at least a
1-in. wall thickness.
2.2 Supplies
and Reagents
1. Rinzl coverslips (18 Â 18 mm, Electron Microscopy Sciences,
Hartfield, PA, USA).
2. Petri dishes (100 mm).
3. Fine-point forceps or tweezers.
4. Standard dissecting scissors.
5. 70% ethanol.
Cryopreservation of Endothelial Monolayers
583
endothelial cells [19]. The monolayer cryopreservation protocol
[19] is described in detail below.
Briefly, our optimized protocol for cryopreservation of endothelial monolayers after culture for 7 days on fibronectin-coated
Rinzl coverslips involves loading of CPA (5% DMSO + 6%
HES + 2% chondroitin sulfate) for 15 min on ice, ice nucleation
at À5
C, cooling to À45
C (at a cooling rate of 1
C/min or
0.2
C/min), and plunging into, and storage in, liquid nitrogen.
Upon rapid thawing of cryopreserved HUVEC monolayers in a
37
C water bath and removal of the CPA by serial dilution, we
obtained an absolute viability of 96.6 Æ 2% when the cooling rate
was 1
C/min and 97.3 Æ 3.2% when the cooling rate was 0.2
C/
min [19]. For porcine corneal endothelial cell monolayers, we
obtained an absolute viability of 91.1 Æ 7.6% when the cooling
rate was 1
C/min and 107.8 Æ 7.8% when the cooling rate was
0.2
C/min to a plunge temperature of À40
C [19] (see Note 1).
2 Materials
2.1 Equipment
1. Analytical balance.
2. Biological safety cabinet.
3. Pipette aid and pipettors.
4. Phase contrast microscope (Labovert, Leitz).
5. Coulter counter or hemocytometer.
6. Centrifuge (Eppendorf 5810R tabletop centrifuge, Hamburg,
Germany) for cell passaging.
7. Programmable methanol cooling bath (FTS Systems, Stone
Ridge, NY, USA).
8. T-type thermocouple and OMB-DAQ-55 data acquisition
module (OMEGA Engineering Inc., Stamford, CT, USA).
9. Water bath.
10. Timer.
11. Liquid nitrogen (LN 2 ) dewar or styrofoam box with at least a
1-in. wall thickness.
2.2 Supplies
and Reagents
1. Rinzl coverslips (18 Â 18 mm, Electron Microscopy Sciences,
Hartfield, PA, USA).
2. Petri dishes (100 mm).
3. Fine-point forceps or tweezers.
4. Standard dissecting scissors.
5. 70% ethanol.
Cryopreservation of Endothelial Monolayers
583