Preventing ice crystal formation is one of the main goals when developing new cryopreservation techniques. Ice crystals cause direct injury to the cells during the freezing and particularly thawing process. The ice is also the main culprit for secondary freezing damage that involves the solutes used in the cryoprotectant solution.
We have been developing alternative cryopreservation solutions that bypass the use of DMSO and reduce as well as minimize the formation of undesirable ice crystals. These solutions are based on naturally-occurring compounds with anti-freeze properties that are biologically compatible with cell membranes. Akron’s family of DMSO-free cryomedia is based on proprietary compositions.
Now, new polymers are emerging as preferred alternatives to proteins as cryoprotectants because of their wider availability, lower cost and ease of handling and preparation.
In a new paper published in Chemical Communications, a group of researchers from the University of Warwick have synthesized a new polymer that limits ice crystal formation in frozen red blood cells as they thaw. The polymer is based on a biomimetic, polyampholite with ice-recrystallization-inhibiting properties.
The authors successfully demonstrated the new polymer’s ability to preserve red blood cells as they thaw with an efficiency comparable to DMSO and glycerol, traditionally preferred cryoprotectants.
The tendency to move toward polyampholytic polymers has, in recent years, emerged with interesting solutions that have challenged the traditionally-accepted DMSO as the cryoprotectant of choice and the notion that other solutions are unable to match its broad efficacy. Polyampholites are, however, not the only polymer compound with cryoprotective properties: companies such as Akron have researched alternative solutions with performance that surpasses DMSO. Though there is still a battle to fight as we learn of the new solutions’ clinical utility, the growing body of work is contributing to the field’s move forward.
Contact us if you would like to enquire about our cell and tissue-specific cryopreservation solutions.
Last week, a new study published in Nature described a promising new approach for the treatment of metabolic disease based on genetically modified pluripotent stem cells.
The high mutation rate in the mitochondrial genome may lead to a number of significant complications, which include neurological, gastrointestinal, cardiac, respiratory, endocrinal and ophthalmological issues and diseases that often have serious, and even fatal, consequences. Avoiding or controlling such mutations has significant medical impact.
The new study described successfully generating genetically-corrected induced pluripotent stem cells (iPSCs) derived from patients with heteroplasmic mutations causing mitochondrial encephalomyopathy, stroke-like episodes and Leigh syndrome. The authors used spontaneous segregation of heteroplasmic mtDNA as well as somatic cell nuclear transfer (SCNT) enabled to generate corrected iPSCs. These genetically “corrected” displayed normal metabolic function compared to that observed in mutant cells.
The study was led by Dr. Shoukhrat Mitalipov at Oregon Health and Sciences University, together with collaborators at the Salk institute for Biological Sciences, the University of California San Diego, the Mayo Clinic and the University of Oxford.
This reprogramming approach is unique and significant in that it successfully generated “healthy” cells from mutated cells in diseased subjects, and represents a new paradigm for the potential therapeutic use of such reprogramming techniques to obtain wild-type mtDNA.
Fibronectin-scaffold composites have emerged as promising three-dimensional substrates for tissue regenerative applications. These structures have shown potential in the regeneration of a variety of tissues. Among those, bone tissue has been of particular interest. The integration of nanotechnology with scaffold fabrication approaches has given rise to new families of structures that allow more thorough integration of biological components that promote tissue repair.
Now, a new paper, titled “Fibronectin immobilization on to robotic-dispensed nanobioactive glass/polycaprolactone scaffolds for bone tissue engineering”, describes the use of robotics, nanotechnology and biomedical engineering to create composite scaffolds for bone regeneration. Moreover, they use the ligand-like properties of fibronectin to improve the attachment of mesenchymal stem cells seeded on the scaffolds.
The authors, led by Dr. Hae-Won Kim at Dankook University in South Korea, described the use of robotics assembly of sol-gel-based glass-PCL scaffolds with immobilized fibronectin which were then used for bone cell proliferation as precursors for osteogenesis. The scaffolds were manufactured by a robotic platform called EZROBO3, in addition to a number of chemical lab complexation steps.
The authors showed that the FN-nBG/PCL scaffolds significantly improved cell responses, including attachment and subsequent cell proliferation of the mesenchymal stem cells seeded on the scaffolds. These effects are dependent on the cell-binding characteristics of fibronectin and are unique to the molecule.
Fibronectin/scaffold composites were previously described in the literature and are emerging as a new paradigm for next-generation three-dimensional tissue regeneration platforms, which are going to require ECM components to generate the kind of in vivo-mimicking responses that have the most medical potential.
Fibronectin is also one of Akron’s leading ECM proteins, which we are now offering in research grade and GMP grade as well with custom MSC-based bioassays. Contact us for information.
Up until now, understanding and investigating the molecular and genetic cues for disease has involved carrying studies in unhealthy individuals, by mobilizing cells that have been affected by the disease. This has been the case for dilated cardiomyopathy (DCM) – a deficiency which affects the heart’s ability to supply blood efficiently.
A new study published in Cell this week, turned this paradigm around by converting induced pluripotent stem cells (iPSCs) from healthy individuals into DCM-impacted cells.
Led by Joseph Wu at Stanford University’s School of Medicine, the authors matched the upregulation of phosphodiesterases (PDEs) 2A and PDE3A that occurs in DCM patient tissue to upregulation in DCM iPSC-CMs. This was important as is further demonstrated the successful reprogramming of iPSCs into DCM cells. The protein TNNT2, which is mutated in DCM patient tissue, was also observed as such in iPSC-derived DCM tissue.
This study is significant not only because it shed further light on the molecular basis for a severe condition such as DCM, but it is remarkable in its use of reprogrammed iPSCs to mimic patient cells. This opens up opportunities for clinical-level studies that will no longer require high-risk studies on sick patients. While this particular study focused on matching patient DCM conditions to genetic cues on iPSC-derived cells, rather than uncovering novel therapeutic outcomes, it is a remarkable first example that such work might soon be possible.
At Akron, we support research into reprogramming of iPSCs by providing media, solutions and new technologies for such investigations. Contact us for more information.
New work from the University of Florida, in collaboration with Cleveland Clinic and the University of California, Berkeley published last week in the Proceedings of the National Academy of Sciences, described a novel microarray device with the ability to deliver combinations of chemotherapeutic drugs to cancer stem cells.
This is based on the principle that a subpopulation of stem cells, termed cancer stem cells, is responsible for initial tumor propagation. However, detecting and working with these cells has been difficult because of their relatively limited numbers.
The authors, led by Benjamin G. Keselowsky, Ph.D., an associate professor in the J. Crayton Pruitt Family Department of Biomedical Engineering, developed a microarray which delivers combinations of chemotherapeutic drugs to small numbers of stem cells captured on the microarray.
The remarkable features are the ability to treat and detect a number of cells that amounts to 6% of what a regular 96-well plate utilizes. The authors demonstrated efficiency by observing and recording responses to chemotherapeutic drugs when colorectal stem cells were grown on the microarray. The cells were taken from a 70-year-old, stage IV cancer patient and a 60-year-old patient with stage III colorectal cancer. The microarrays were seeded with only approximately 200 cells per culture group and results were in agreement within 14% error between test groups.
While the results reported in this manuscript came from only one cell type, the system opens up the opportunity for more rapid and sensitive cancer treatment.
The paper, titled “Drug-eluting microarrays to identify effective chemotherapeutic combinations targeting patient-derived cancer stem cells,” can be accessed here.
To support further research into oncology, and learn more on stem cell media, culture conditions and custom growth substrates, contact us.
Last week, Nature dedicated an technology editorial to new advances in tissue engineering, more specifically the “organs-grown-in-a-lab” sub-field. Titled Tissue engineering: Organs from the lab, the article proclaimed, “Engineered tissues are starting to allow incisive experiments and even replacement therapies,” before concluding that new discoveries and advancements in tissue engineering not only offer a “better understanding of the basis of disease” but also a real potential to “cure those diseases.”
This comes on the heels of some remarkable new developments. MIT associate professor of mechanical engineering Xuanhe Zhao and colleagues at MIT, Duke University, and Columbia University described, in the journal Advanced Materials, the development of new hydrogels that are not only tough, but soft and “stretchable,” which can be assembled with approaches that do not require harsh chemicals. Not only that, but the hydrogels can be assembled into a variety of 3D structures by 3D printing, which opens up possibilities of their use as cell delivery carriers as well as scaffolds for expansion of stem cells. Indeed, the new materials are benign enough to synthesize together with living cells, such as stem cells. The authors are currently focused on improving the resolution of the printer, which is currently limited to details about 500 micrometers in size.
This comes in addition to a new paper, published in Nature Materials, from Dr. Dino di Carlo’s lab at UCLA, which describes a new injectable polymer gel for the rapid treatment of wounds.
The gel uses a cluster of microscopic synthetic polymer spheres, which produces MAP (microporous annealed particles) gel, which fills the wound and facilitates the growth of new tissue. Eventually the body degrades the spheres, leaving just the newly grown tissue. Cells are encouraged to migrate into the microporous gel and proliferate, and in doing so, encourage the assembly of new tissue.
New biomaterials will be they key to achieving biocompatible and effective tissue replacement and regeneration, and advances such as these are at the forefront of systems development toward effective biomedical therapies in regenerative medicine.
Improving cryopreservations strategies and developing optimal formulations of cryoprotectants is a high priority for the cell therapy and medical fields. A recent study from the Royal Free Hospital in London, UK investigated the toxicity associated with the exposure of cord blood samples to DMSO. The authors carried out cell viability and in vitro functional assays in fresh and post-thaw cord blood samples, and determined that the optimal concentration of DMSO for cryopreserved cord blood is in the 7.5 – 10% range, while the maximum exposure time should be limited to <1 h prior to freezing and 30 min post-thaw.
Elsewhere, new strategies for cryopreservation have appeared. The lab of Francisco del Monte at the Materials Science Institute of Madrid described the development, in the journal ACS Applied Materials and Interfaces, of liquid marbles encapsulating fibroblasts that are used for cryopreserving the encapsulated cells. Liquid marbles are spherical constructs – they are typically described as droplets – made of polymeric materials surrounding an aqueous core. They have been used for many applications. In the paper, the authors encapsulated murine L929 fibroblasts inside liquid marbles made up of poly(tetrafluoroethylene) and found that the cells were well cryopreserved in such a construct adhesion, morphology, viability, proliferation, and cell cycle. More studies are needed to further understand and optimize the utility of such processes, but this is one of the earliest examples of such a system.
Encapsulation was also employed in a new study on cryopreservation by a Jose Luis Pedraz’ lab at the University of the Basque Country in Spain. The authors tested a number of cryoprotectant solutions combining DMSO, glycerol and trehalose on encapsulated mesenchymal stem cells genetically modified to secrete erythropoeitin. Cells were encapsulated in multi-cell particles and were cryopreserved either with DMSO, glycerol or trehalose alone or a combination of cryoprotectants. The authors found that DMSO at a concentration of 10% displayed the best viability and erythropoietin secretion profile compared to the other cryoprotectant solutions. This is not, however, meant to exclude other compositions as being suitable for further development and optimization, considering the nature of this study was limited.
Some of these studies highlight that encapsulation, but more generally, novel approaches for cryopreservation, may hold keys to new solutions for the protection of hard-to-preserve cells for clinical use.
For more information about solutions for cryopreservation using DMSO as well as novel DMSO-free approaches, get in touch with us.