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Visualization of Physical Effects during Cryopreservation using DP6 in Combination with Synthetic Ice Modulators (SIMs)

 

The first cryomacroscope was invented in 2003 and has been developed ever since at the BTTL. Cryomacroscopy has been demonstrated as an effective means to visualize physical effects during typical cryopreservation protocols. Five generations of the cryomacroscope have been presented over the past 15 years, for various applications, ranging from measurements of thermophysical properties to monitoring of large-scale cryopreservation, with the Polarized-Light Scanning Cryomacroscope as the most advanced development thus far. Presented below are examples of cryomacroscopy videos from experiments conducted to study Synthetic Ice Modulators (SIMs) on blood vessels and ovaries (3rd generation cryomacroscopy).

The cryoprotective agent (CPA) cocktail used for blood vessel experiments is DP6 and several of its derivatives. DP6 is a mixture of 3M dimethyl sulfoxide (DMSO) and 3M propylene glycol in a vehicle solution (Unisol in most current experiments). For the cryopreservation experiments on ovaries, M22 is used as the CPA cocktail, which is a complex cocktail containing five permeating ingredients: DMSO (22.3%), formamide (12.9%), ethylene glycol (16.8%), N-methylformamide (3%), and 3-methoxy-1,2-propanedio (4%); and three non-permeating ingredients (also known as ice blockers, or SIBs): polyvinyl pyrolidone K12 (2.8%), X-1000 (1%) and Z-1000 (1%). SIMs are compounds that influence the nucleation and growth of ice crystals by various purported mechanisms, where SIBs are examples of SIMs used in the cocktail M22.

The cryoprotocols displayed below start with a faster cooling rate down to -100°C, to suppress crystallization, followed by slower cooling down to the storage temperature, to minimize thermo-mechanical stress. A similar strategy in a reverse order is employed during rewarming, where a slower rewarming from storage temperature to -100°C helps in minimizing thermomechanical stress followed by faster rewarming to outrun rewarming phase crystallization.

 

 

 

Pig Femoral Artery undergoing cryopreservation using 10 ml of DP6 + X1000 + Z1000.
Highlighted effects:
Crystals begin to grow on the artery (02:57)
Hexagonal crystallization in the specimen (04:07)
Dense crystallization makes the specimen opaque (06:35)
Melting is observed on the periphery during rewarming (13:51)
Rewarming phase crystallization can be observed (14:11)

 

 

 

 

Pig Femoral Artery undergoing cryopreservation using 10 ml of DP6 + X1000 + Z1000 + 0.175M Sucrose.
Highlighted effects:
Crystallization on the top surface of the specimen (05:11)
The rest of the specimen vitrifies (12:08)
The vitrified CPA at the periphery begins to melt (12:19)
Rewarming phase crystallization makes the surface opaque (13:45)

 

 

 

 

Pig Femoral Artery undergoing cryopreservation using 10 ml of DP6 + 0.175M Sucrose.
Highlighted effects:
The video becomes blurry due to cavity formation at the top surface (06:59)
The specimen vitrifies as a clear glassy solid (10:22)
Rewarming phase crystallization makes the surface opaque (13:48)

 

 

 

 

Rabbit jugular vein undergoing cryopreservation using 4 ml of DP6 + 0.5M Sucrose + Unisol.
Highlighted effects:
The specimen vitrifies into a glassy solid (09:47)
High thermomechanical stress causes fracture formation (10:04)
As rewarming progresses, the viscosity of the specimen increases and fractures disappear (13:14)
Rewarming phase crystallization makes the surface opaque (13:36)

 

 

 

 

Ovary undergoing cryopreservation protocol using M22 as the CPA.
Highlighted effects:
Crystallization begins to develop on the ovaries (01:47)
The top surface of the specimen begins to crystallize and become opaque (03:17)
Rewarming ends and the crystals melt (08:50)

 

 

 

 

Rabbit jugular vein undergoing cryopreservation in 10 ml of DP6 + 0.175M Sucrose + Unisol.
Highlighted effects:
Crystallization begins to develop at the top surface of the specimen (04:38)
The top surface of the specimen becomes completely opaque (05:13)
Crystals melt and the specimen becomes clear (14:43)

 

 

 

 

Pig Femoral Artery undergoing cryopreservation using 10 ml of DP6 + 0.175M Sucrose. Highlighted effects:
The artery specimen begins to crystallize (02:51)
The CPA around the artery vitrifies (10:03)
The specimen becomes opaque due to rewarming phase crystallization (13:51)



 

 

Pig Femoral Artery undergoing cryopreservation protocol using 10 ml of DP7 + 0.6M Sucrose. Highlighted effects:
The specimen vitrifies with no visible crystallization (10:11)
The specimen becomes opaque due to crystallization during rewarming (13:49)

 

 

 

 

Rabbit jugular vein undergoing cryopreservation using 10 ml of DP6 as CPA + 0.5M Sucrose + Unisol. Highlighted effects:
The specimen vitrifies with no visible crystallization (09:56)
Some crystallization is seen on the top surface of the specimen (14:08)
The specimen becomes clear at the end of rewarming protocol (14:37)

 

 

 

 

Pig Femoral Artery undergoing cryopreservation using 10 ml of DP6 + 0.6M Sucrose. Highlighted effects:
Crystals can be seen growing on the artery specimen (03:04)
The CPA around the specimen vitrifies into a glassy solid (09:44)
Rewarming phase crystallization causes the specimen to become opaque (13:30)

 

 

 

 

Pig Femoral Artery undergoing cryopreservation using 10 ml of DP6 + 0.6M Sucrose. Highlighted effects:
The specimen vitrifies with no visible crystallization (09:51)
The specimen becomes opaque due to crystallization during rewarming (14:06)

 

 

   

 

Selected Publications:

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Wowk, B.G., Fahy, G.M, Ahmedyar, S., Taylor, M.J., Rabin, Y. (2018): Vitrification tendency and stability of DP6 vitrification solutions for complex tissue cryopreservation, Cryobiology, 82:70-77, PubMed, HHS Public Access, ScienceDirect

 

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Solanki, P.K., Rabin, Y. (2018): Analysis of polarized-light effects in glass-promoting solutions with applications to cryopreservation and organ banking, PLoS ONE, 13(6): e0199155 PubMed, HHS Public Access, plos.org

 

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Ehrlich, L.E., Malen, J.A., Rabin, Y. (2016): Thermal conductivity of the cryoprotective cocktail DP6 in cryogenic temperatures, in the presence and absence of synthetic ice modulators, Cryobiology, 73(2):196-202 PubMed, HHS Public Access, ScienceDirect

 

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Feig, J.S.G., Solanki, P.K., Eisenberg, D.P., Rabin, Y. (2016): Polarized light scanning cryomacroscopy, Part II: thermal modeling and analysis of experimental observations, Cryobiology, 73(2):272-281 PubMed, HHS PublicAccess, ScienceDirect

 

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Feig, J.S.G., Eisenberg, D.P., Rabin, Y. (2016): Polarized light scanning cryomacroscopy, Part I: Experimental apparatus and observations of vitrification, Crystallization, and Photoelasticity Effects, Cryobiology, 73(2):261-71 PubMed, HHS Public Access, ScienceDirect

 

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Feig, J.S.G., Rabin, Y. (2014): The scanning cryomacroscope with applications to cryopreservation – a device prototype, Cryogenics, 62:118–128 HHS Public Access, ScienceDirect

 

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Feig, J.S.G., Rabin, Y. (2013): Integration of polarized light into scanning cryomacroscopy. CRYO2013-the 50 th Annual Meeting of the Society for Cryobiology, N. Bethesda, DC (July 28-31), Cryobiology, 67(3):399-400 ScienceDirect

 

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Rabin, Y., Taylor, M.J., Feig, J.S.G., Baicu, S., Chen, Z. (2013): A new cryomacroscope device (Type III) for visualization of physical events in cryopreservation with applications to vitrification and synthetic ice modulators, Cryobiology 67(3):264-73 PubMed, HHS Public Access, ScienceDirect

 

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Rabin, Y., Feig, J.S.G., Williams, A.C., Lin, C.C., Thaokar, C. (2012): Cryomacroscopy in 3D: a device prototype for the study of cryopreservation. ASME 2012 Summer Bioengineering Conference - SBC 2012, Fajardo, Puerto Rico, USA (June 20-23) ASME Digital Collection, BTTL Depository

 

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Baicu, S., Taylor, M.J., Chen, Z., Rabin, Y. (2008): Cryopreservation of carotid artery segments via vitrification subject to marginal thermal conditions: Correlation of freezing visualization with functional recovery. Cryobiology, 57(1):1-8 PubMed, HHS Public Access, ScienceDirect, BTTL Depository

 

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Steif, P.S., Palastro, M.C, Rabin, Y. (2008): Continuum mechanics analysis of fracture progression in the vitrified cryoprotective agent DP6. ASME Biomechanical Engineering, 130(2):021006 PubMed, HHS Public Access, ASME Digital Collection

 

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Rabin, Y., Steif, P.S., Hess, K.C., Jimenez-Rios, J.L., Palastro, M.C. (2006): Fracture formation in vitrified thin films of cryoprotectants. Cryobiology, 53:75-95 PubMed, HHS Public Access, ScienceDirect, BTTL Depository

 

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Baicu, S., Taylor, M.J., Chen, Z., Rabin, Y. (2006): Vitrification of carotid artery segments: An integrated study of thermophysical events and functional recovery towards scale-up for clinical applications. Cell Preservation Technology, 4(4):236-244 PubMed, HHS Public Access, BTTL Depository

 

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Rabin, Y., Steif, P.S. (2006): Solid mechanics aspect of cryobiology, In: Advances in Biopreservation (Baust, J.G., and Baust J.M., Eds.), CRC Taylor & Francis, Chap. 13, pp. 359-382

 

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Rabin, Y., Taylor, M.J., Walsh, J.R., Baicu, S., Steif, P.S. (2005): Cryomacroscopy of vitrification, Part I: A prototype and experimental observations on the cocktails VS55 and DP6. Cell Preservation Technology, 3(3):169-183 PubMed, HHS Public Access, BTTL Depository

 

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Steif, P.S., Palastro, M., Wen, C.R., Baicu, S., Taylor, M.J., Rabin, Y. (2005): Cryomacroscopy of vitrification, Part II: Experimental observations and analysis of fracture formation in vitrified VS55 and DP6. Cell Preservation Technology, 3(3):184-200 PubMed, HHS Public Access, BTTL Depository

 

 

Acknowledgments:

•       National Heart Lung and Blood Institute (NHLBI) Grant R01HL127618

 

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