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A Novel Protocol Permitting the Use of Frozen Cell Cultures on Low Earth Orbit

L. S. Kidder
L. S. Kidder
, 
L. Zea
L. Zea
, 
SM Countryman
SM Countryman
, 
L. S. Stodieck
L. S. Stodieck
 and 
B. E. Hammer
B. E. Hammer
   | Jun 14, 2020
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Article Category: Research Note
Published Online: Jun 14, 2020
Page range: 25 - 30
DOI: https://doi.org/10.2478/gsr-2020-0003
Keywords
© 2020 L. S. Kidder et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

(A) Engineering drawing of BioCell and (B) BioCell with frozen cell culture/media. The needle-less Luer connectors allow the exchange of media.
(A) Engineering drawing of BioCell and (B) BioCell with frozen cell culture/media. The needle-less Luer connectors allow the exchange of media.

Figure 2

Prototype aluminum block designed to defrost frozen BioCells on orbit. Both sides of this block were heated to 43°C and positioned on the frozen BioCell. It was ultimately determined that aluminum was not effective in rapidly defrosting frozen cultures.
Prototype aluminum block designed to defrost frozen BioCells on orbit. Both sides of this block were heated to 43°C and positioned on the frozen BioCell. It was ultimately determined that aluminum was not effective in rapidly defrosting frozen cultures.

Figure 3

Metal enclosure housing BioCells (PHAB). Note the vents at the top of the enclosure allowing gas exchange while it is maintained in the 37°C, 5% CO2 incubator.
Metal enclosure housing BioCells (PHAB). Note the vents at the top of the enclosure allowing gas exchange while it is maintained in the 37°C, 5% CO2 incubator.

Figure 4

BioCell thaw system. The bag labeled “Reservoir” contains water heated to 43°C. The frozen BioCell is placed in the bag labeled “Thawing Chamber” which is then sealed. Air is evacuated, and a large clip is opened between the two bags, and warm water pushed in. The thawing bag is again sealed. With rocking/kneading, the BioCell is defrosted within 2–3 min. The clip between the two bags is then opened, and water is pushed back into the reservoir bag.
BioCell thaw system. The bag labeled “Reservoir” contains water heated to 43°C. The frozen BioCell is placed in the bag labeled “Thawing Chamber” which is then sealed. Air is evacuated, and a large clip is opened between the two bags, and warm water pushed in. The thawing bag is again sealed. With rocking/kneading, the BioCell is defrosted within 2–3 min. The clip between the two bags is then opened, and water is pushed back into the reservoir bag.

Figure 5

Time course for thawing of BioCell from −80°C; n = 4. The internal temperature of the BioCell was determined using a resistance temperature detector (RTD) (Omega HSRTD-3-100-A-80-E, Omega Engineering, Inc., Norwalk, CT) interfaced to LabVIEW 2018 (National Instruments, Austin, TX) through a customized virtual instrument and sampled at 1 s increments. (A) BioCell exposed to air at 20°C. (B) BioCell submerged in 1,000 ml water bath (23 cm × 28 cm) initially at 43°C. Water depth is 19 mm before submerging BioCell. The standard deviation is higher because the physical location of the RTD in a frozen BioCell is variable and within a few millimeters of the BioCell membrane. During the thawing process, the BioCell is mechanically agitated to ensure the maximum convection/heat transfer occurs between the BioCell and bath. When an RTD is close to the BioCell membrane, which is in contact with the water bath, it will warm faster than an RTD further from the membrane. (C) Frozen media changing to liquid media. (D) Cell media in liquid state approaches bath temperature. Error bars are displayed every 10 s for the BioCell and bath but are not visible due to when the standard error is small, for example, at 150 s bath temperature is 37.2° ± 0.3°C and BioCell temperature is −3.6° ± 2.6°C.
Time course for thawing of BioCell from −80°C; n = 4. The internal temperature of the BioCell was determined using a resistance temperature detector (RTD) (Omega HSRTD-3-100-A-80-E, Omega Engineering, Inc., Norwalk, CT) interfaced to LabVIEW 2018 (National Instruments, Austin, TX) through a customized virtual instrument and sampled at 1 s increments. (A) BioCell exposed to air at 20°C. (B) BioCell submerged in 1,000 ml water bath (23 cm × 28 cm) initially at 43°C. Water depth is 19 mm before submerging BioCell. The standard deviation is higher because the physical location of the RTD in a frozen BioCell is variable and within a few millimeters of the BioCell membrane. During the thawing process, the BioCell is mechanically agitated to ensure the maximum convection/heat transfer occurs between the BioCell and bath. When an RTD is close to the BioCell membrane, which is in contact with the water bath, it will warm faster than an RTD further from the membrane. (C) Frozen media changing to liquid media. (D) Cell media in liquid state approaches bath temperature. Error bars are displayed every 10 s for the BioCell and bath but are not visible due to when the standard error is small, for example, at 150 s bath temperature is 37.2° ± 0.3°C and BioCell temperature is −3.6° ± 2.6°C.

Figure 6

Photomicrograph of MC3T3 osteoblastic cells post-defrost in situ (100×). Cells survived cryopreservation and proliferated normally.
Photomicrograph of MC3T3 osteoblastic cells post-defrost in situ (100×). Cells survived cryopreservation and proliferated normally.

Quality and quantity of RNA recovered from flight cultures as determined by UV spectroscopy.

ID~ Quality (A260 λ/A280 λ)Total quantity (μg)
SN1 – D2.1321.04
SN1 – E2.0512.76
SN1 – F2.1019.43
SN2 – D2.064.81
SN2 – E2.108.86
SN2 – F–0.00
SN3 – D–0.00
SN3 – E2.1018.22
SN3 – F2.108.25
SN4 – D2.002.24
SN4 – E2.1239.41
SN4 – F2.1439.49
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