The introduction of poly(dimethylsiloxane) (PDMS) and soft lithography in the 90's has revolutionized the field of microfluidics by almost eliminating the need for a clean-room environment for device fabrication. More recently, 3D printing has been introduced to fabricate molds for soft lithography, the only step for which a clean-room environment is still often necessary, to further support the rapid prototyping of PDMS microfluidic devices. However, toxicity of most of the commercial 3D printing resins has been established, and little is known regarding the potential for 3D printed molds to leak components into the PDMS that would, in turn, hamper cells and/or tissues cultured in the devices. In the present study, we investigated if 3D printed molds produced by stereolithography can leach components into PDMS, and compared 3D printed molds to their more conventional SU-8 counterparts. Different leachates were detected in aqueous solutions incubated in the resulting PDMS devices prepared from widely used PDMS pre-polymer:curing agent ratios (10:1, 15:1 and 20:1), and these leachates were identified as originating from resins and catalyst substances. Next, we explored the possibility to culture cells and tissues in these PDMS devices produced from 3D printed molds and after proper device washing and conditioning. Importantly, we demonstrated that the resulting PDMS devices supported physiological cultures of HeLa cells and ovarian tissues in vitro, with superior outcomes than static conventional cultures. Organ-on-a-chip models, by providing an in vivo-like environment, show great potential to advance our understanding of tissue development, physiology, and pathology 1. However, the spread of these highly promising platforms out of specialized microfluidic laboratories is hindered by essential technical challenges. The fabrication of these devices requires having access to dedicated infrastructure such as clean-room facilities with specialized equipment, which is absent in most biological laboratories 1. Novel fabrication processes have been developed to support the rapid prototyping of microfluidic devices, including soft-lithography using PDMS (polydimethylsiloxane) 2,3. PDMS is a transparent, flexible, gas-permeable, fairly inexpensive and rapidly prototyped elastomeric material, which has now become ubiquitous in the field of microfluidics 2,3. PDMS devices are commonly fabricated using molds based on SU-8 (Microchem Corp.), which is a negative epoxy-based photoresist 4. However, the fabrication of SU-8 molds uses lithography and includes multiple steps, it altogether takes several hours per fabrication cycle, and still requires dedicated training and access to a clean-room facility 5,6. As a new revolution in the field of microfluidics, 3D printing has more recently been introduced 7. Initially the use of 3D printing was limited by the price of the printers, their low resolution, the roughness of the 3D printed structures, and the toxicity of the resins 8-10. Photopolymerization-based techniques, such as dig...
The ability to spur growth of early stage gametic cells recovered from neonates could lead to significant advances in rescuing the genomes of rare genotypes or endangered species that die unexpectedly. The purpose of this study was to determine, for the first time, the ability of two substantially different cryopreservation approaches, slow freezing versus vitrification, to preserve testicular tissue of the neonatal sheep and subsequently allow initiation of spermatogenesis post-xenografting. Testis tissue from four lambs (3-5 wk old) was processed and then untreated or subjected to slow freezing or vitrification. Tissue pieces (fresh, n = 214; slow freezing, then thawing, n = 196; vitrification, then warming, n = 139) were placed subcutaneously under the dorsal skin of SCID mice and then grafts recovered and evaluated 17 wk later. Grafts from fresh and slow frozen tissue contained the most advanced stages of spermatogenesis, including normal tubule architecture with elongating spermatids in ~1% (fresh) and ~10% (slow frozen) of tubules. Fewer than 2% of seminiferous tubules advanced to the primary spermatocyte stage in xenografts derived from vitrified tissue. Results demonstrate that slow freezing of neonatal lamb testes was far superior to vitrification in preserving cellular integrity and function after xenografting, including allowing ~10% of tubules to retain the capacity to resume spermatogenesis and yield mature spermatozoa. Although a first for any ruminant species, findings also illustrate the importance of preemptive studies that examine cryo-sensitivity of testicular tissue before attempting this type of male fertility preservation on a large scale.
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