Capillary assisted vitrification processes and devices
Inventors
MOHANTY, PRAVANSU S. • Chakraborty, Nilay
Assignees
Somnio Global Holdings LLC • Ambient Biosciences Inc
Publication Number
US-10568318-B2
Publication Date
2020-02-25
Expiration Date
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Abstract
Disclosed are devices and methods for non-cryogenic vitrification of biological materials that include the steps of providing one or more capillary channels of which a first opening is operably in contact with a moisture containing vitrification mixture made of a biological material and a vitrification agent. The capillary absorbs and transports the moisture to the second opening through capillary action, and the moisture is subsequently evaporated into a surrounding low humidity atmosphere until the vitrification mixture enters into a vitrified state.
Core Innovation
The invention provides devices and methods for non-cryogenic vitrification of biological materials by capillary assisted fast drying. The disclosure describes providing one or more capillary channels of which a first opening is operably in contact with a moisture containing vitrification mixture made of a biological material and a vitrification agent, the capillary absorbing and transporting the moisture to the second opening through capillary action, and the moisture subsequently evaporated into a surrounding low humidity atmosphere until the vitrification mixture enters into a vitrified or glassy state. The disclosure further provides a vitrification device comprising a receptacle with walls containing a plurality of capillary channels and an enclosure in gaseous communication with the second openings of the capillary channels, wherein the pressure, temperature and humidity within the enclosure can be controlled.
The invention addresses limitations of prior art vitrification and desiccation methods that require extremely high and potentially toxic concentrations of cryoprotectants and that suffer from slow, non-uniform evaporative drying which forms a glassy skin and prevents further desiccation. The disclosure identifies a need for a fast and practical desiccation technique to achieve very low and uniform final moisture levels while maintaining material viability, and proposes capillary assisted drying combined with appropriate vitrification compositions (for example trehalose, glycerol and ionic buffer containing large organic ions such as choline and betine) to preserve structural integrity during fast drying and enable storage above cryogenic temperatures.
Claims Coverage
The specification provides one independent process claim. The independent claim recites five main inventive features corresponding to steps (a)–(e).
Membranes with plurality of contiguous capillary channels
Providing a first membrane and a second membrane, the first membrane, the second membrane or both comprising a plurality of contiguous capillary channels, said capillary channels having a first opening and a second opening.
Vitrification mixture comprising biological materials and a vitrification medium
Providing a vitrification mixture, said vitrification mixture comprising one or more biological materials and a vitrification medium.
Dual-sided membrane contact with the vitrification mixture
Contacting said first membrane and said second membrane with said vitrification mixture such that the first membrane contacts a first surface of the vitrification mixture and the second membrane contacts a second surface of the vitrification mixture, the first membrane and second membrane contacting the vitrification mixture such that the first opening of said capillary channels is operably in contact with said vitrification mixture.
Second openings in direct communication with lower-humidity atmosphere
Wherein said second openings of said capillary channels are directly operably in communication with a surrounding atmosphere having humidity below that of said vitrification mixture.
Capillary-action desiccation above cryogenic temperature to glassy state
Desiccating away the said vitrification mixture by capillary action above cryogenic temperature until the said vitrification mixture enters into a glassy state.
The independent claim defines a process combining (i) membranes comprising contiguous capillary channels, (ii) a vitrification mixture of biological material and vitrification medium, (iii) dual-sided membrane contact with the mixture, (iv) exposure of capillary second openings to a lower-humidity atmosphere, and (v) capillary-action desiccation above cryogenic temperature until vitrification (glassy state) is achieved.
Stated Advantages
Fast and uniform moisture removal during non-cryogenic vitrification of biological materials.
Preservation of structural integrity (physiological and/or molecular) of biological material during fast drying using a preferred vitrification composition comprising trehalose, glycerol and an ionic buffer containing large organic ions such as choline and betine.
Ability to achieve very low and uniform final moisture levels across the sample, overcoming glassy-skin induced non-uniformity of conventional sessile droplet desiccation.
Enables vitrification above cryogenic temperatures and facilitates long-term storage of vitrified biological materials, including storage in a protective enclosure impervious to water and air at temperatures between −196° C. to +60° C.
Capability to vitrify larger sample volumes without requiring ultra-thin film formats and to retain cell membrane integrity and protein activity (for example insulin) after desiccation and rehydration.
Documented Applications
Preservation and vitrification of biological materials including proteins, cells, tissues, organs, cell-based constructs, reproductive cells (sperm, spermatocytes, oocytes, embryos, germinal vesicles), whole blood components, viruses, bacteria, algae, fungi, liposomes, enzymes.
Vitrification and storage of mammalian cells including examples provided such as Chinese hamster ovary (CHO) cells and mouse oocytes.
Stabilization and retention of activity for protein therapeutics exemplified by insulin following capillary assisted vitrification and rehydration.
Long-term storage and transportation of vitrified biological materials sealed in protective enclosures, including storage between −196° C. to +60° C. and potential ambient storage without a cold chain for some samples.
Use of a non-cryogenic vitrification device comprising receptacle(s) with capillary channels and an enclosure with controlled pressure, temperature and humidity; configurations include multi-cavity devices and embodiments with internal capillary pipes/wicks and wearable device possibilities.
Application to vaccines and proteinaceous materials explicitly mentioned as examples including interleukin‑1, interleukin‑2, tetanus and hepatitis vaccines.
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