Since cell therapy has become a reliable therapeutic tool, the collection and transportation of patient cells has become crucial steps in all treatments, because most cell therapies require cell manipulations that can only be done by expert laboratories, for instance growing or genetic modification (1). As a consequence, sample collection is performed in one place and the cells are shipped to the process laboratories,which could be very distant in some cases, even in different countries or continents. After processing, the cells are shipped again to the hospital or medical facility where the patient will be treated. All these steps involve time in which cells are alive, exposed to different environments, and enduring exposure in harsh media and additives, therefore degrading in function and vitality. The goal in these methods is to deliver the maximum amount of viable cells to the patient, and this requires slowing down all cellular activities that involve apoptosis and death, throughout the shipping period, and before administration.Traditionally that has been achieved by deep freezing the cells (cryopreservation), following procedures that engage the use of liquid nitrogen and mixing the media with precise amounts of DMSO. These or similar additives are known to be cytotoxic at normal temperature (22OC +), and residual traces of it produce side effects to the patients. Moreover, transportation of frozen cells requires expensive, specialized containers, to hold very low temperatures (at least 70OC below zero) throughout the traveling time. This is normally achieved with large dewars or cold shippers filled with dry ice that maintain the frozen material at temperatures below -60OC, though if the cells are warmed the DMSO will kill all of them in a few minutes. Because of the cells’ temperature sensitivity under these conditions, cell therapies require complicated and expensive temperature monitors, real-time reporting and software to alert in the event of temperature changes. This model is a disaster. Other new concepts involve cell encapsulation in hydrogel bubbles, which are composed by biological molecules. This technology requires processing prior to shipping and chemical treatment to dissolve the carrier and may introduce immune active responses in the patient. Therefore, these methods introduce multiple complicated steps that are totally unnecessary. Current “alternative” procedures provide no practical advantage to cryo-shipping. The circuit from the patient,through processing, and back to the patient goes through these steps:
1. Patient cell extraction.
2. Cell transfer to special containers.
3. Cell preparation for freezing (with cell death), or encapsulation.
4. Freezing cells (with cell death), or encapsulating.
5. Cryo transportation (expensive and with heavy risk of total loss), or gel transportation, theoretically at ambient temperature (?) with additional additives and processing
6. Cell thawing (with cell death), or cell de-encapsulation (unknown cell death and risks).
7. Cell processing,
8. Cell transfer to special containers.
9. Cell preparation for freezing (with cell death), or encapsulation.
10. Freezing cells (with cell death), or encapsulating.
11. Cryo transportation (expensive and with heavy risk of total loss), or gel transportation, theoretically at ambient temperature (?)with additional additives and processing
12. Cell thawing (with cell death), or cell de-encapsulation (unknown cell death).
13. Cell washing.
14. Finally cells are used in the patient’s treatment.
In summary, all these processes require many transfers of cells to various containers, with the consequent risk of contamination and cell loses, six required steps each with cell loses risk, and two steps of high risk of losing all cells.This is a very inefficient, time-consuming, risky and expensive process. This is why there is an intense search for alternative methods that are less complicated, more robust and at lower cost. Currently, the Achilles heel of this process is the cryopreservation. This is the target to be changed by something safer, simple, easier, and less expensive.The ultimate solution has already been provided by commercial applications of culturing cells, where methods have been developed to increase the production of recombinant proteins in mammalian cell cultures by reducing rampant metabolism and cell growth. Cell cycle speed reduction increases the proportion of cells in the G1-phase, which subsequently increases their specific protein productivity (1).
This speed reduction can be induced through cryostatic agents such as Sodium butyrate (NaBu) (2), DMSO (3), over-expression of cell cycle inhibitory proteins p21Cip1 (4) or through control of the modification of the culture environment including reduced O2 levels and “mild hypothermia” (5).
While mild hypothermia is an easy application in suspension cell cultures in industrial tank bioreactors, there are several difficulties to be applied in small culture flasks and dishes where dehydration is a limiting factor. Incubation at 28 OC or 30 OC, necessary for inducing cell cycle speed reduction, is not high enough to obtain the level of environmental relative humidity required for blocking the media evaporation.
A classical cellular response to hypoxia is a pause of growth (6). Hypoxia-induced cell growth arrest varies across cell types but is likely an essential aspect of in-vivo tissue repair control in the response to wounding and injury. A major
constituent of the hypoxic response is the activation of the hypoxia-inducible factor 1 (HIF-1) transcription factor through the involvement of the tumor suppressor protein p53 (7). Deep hypoxia (0.5 % O2 = 1.3 mmHg) can control cell proliferation through growth speed reduction. In cancer cells, hypoxia can induce apoptosis via the p53 pathway and p27 expression (8). However, environmental mild hypoxic (20 to 30 mmHg) can induce cell cycle delay at the G1/S interface without any alteration in their long-term viability or function.
In addition acidosis also has a major role in controlling the cells growth through the key functions of p53 (9). Below pH 7 glucose consumption (10) is reduced in cultured cells and cell cycle become slower, up to 0.2 times the cell cycle time at pH 7.4, extending by 500% the doubling time (example: from 20h to 100h, or from 1 day to 5 days).
In conclusion, combining these three environmental factors (mild hypoxia, mild hypothermia and mild acidosis) cells can be successfully maintain in a dormant state for many days. This phenomenon allows cells to be transported across the world without cryo processing, hydrogel suspension, media additives or any processing at all, and with minimal risk. In addition, cell pausing (stasis), totally avoids dangerous complex and expensive procedures and expensive containers and monitoring equipment and software, while offering a much more robust storage and shipping process. This mimics in-vivo phenomenon of normal tissue repair and remodeling and is totally natural.
The PetakaG3™ bioreactor perfectly maintains these three environmental traits controlled by:
A self-contained proprietary humidity diffusion system which prevents dehydration for weeks and even months at any temperature maintained in >10% relative humidity. Therefore, cells can be maintained in PetakaG3™ at 20 OC or 30 OC, for weeks at any temperature, and 10% relative humidity environment,with no risk of accidental dehydration, achieving al level of hypothermia compatible with cell pausing for shipping.
A self-contained, proprietary oxygen diffusion control system that automatically regulates the media dissolved partial pressure of O2. This system in equilibrium with the cell respiratory level maintains the dissolved oxygen partial pressure within the lowest physiologic oxygen levels (between 10 and 35 mmHg), thus activating the cell growth reduction by doubling time extension up to 200 hours in average. Therefore cells can be maintained in regular media at room temperature for days, even weeks with minimalcell loss and no long-term damage.
A CO2diffusion controlled system which maintains buffer stability at physiologic pH, adjusted for cell dormancy, and extremely long doubling times. The Petaka G3™ is like a perfectly functioning portable incubator, in the form of an ultra low-cost disposable.
Therefore, cells can be held in PetakaG3™ at 20 OC or 30 OC, achieving a level of hypothermia compatible with mild reversible cell pausing.Therefore, cells can be transported in the PetakaG3™ within 10 OC and 30 OC with no environmental risks, for periods of time compatible with the modern transportation systems, between any two points of the world, reducing the cost of logistics by 80% and reducing by 90% the risk of cell loses and contamination, therefore shrinking the entire circuit to:
1. Patient cell extraction.
2. Cell transfer to Petaka G3™.
3. Petaka G3™ transportation at ambient temperature.
4. Cell processing.
5. Cell transfer to Petaka G3™.
6. Petaka G3™ transportation at ambient temperature.
7. Finally cells are used in the patient’s treatment.
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REFERENCES:
https://aabme.asme.org/posts/industry-confronts-challenge-of-shipping-cells-for-therapy
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Louis M, Rosato RR, Brault L, Osbild S, Battaglia E, Yang X-H, et al. The histone deacetylase inhibitor sodium butyrate induces breast cancer cell apoptosis through diverse cytotoxic actions including glutathione depletion and oxidative stress. Int. J. Oncol. 2004 Dec;25(6):1701–11.
Fiore M, Zanier R, Degrassi F. Reversible G1 arrest by dimethyl sulfoxide as a new method to synchronize Chinese hamster cells. Mutagenesis. 2002;17(5):419–24.
Wang Z, Lee H-J, Chai Y, Hu H, Wang L, Zhang Y, et al. Persistent p21Cip1 induction mediates G(1) cell cycle arrest by methylseleninic acid in DU145 prostate cancer cells. Curr Cancer Drug Targets. 2010 May;10(3):307–18.
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