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Device and method for freeze drying biological samples

A biological sample, drying technology, applied in the direction of drying solid materials, drying gas layout, preservation of human or animal bodies, etc., can solve the problems of irreversible loss of storage tank failure samples, expensive, troublesome transportation, etc.

Inactive Publication Date: 2020-10-30
安全生殖公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this effective method of cryopreservation is expensive
Storing cryopreserved samples under liquid nitrogen (LN) has high maintenance costs and requires dedicated dedicated facilities and highly trained staff
Plus, shipping is cumbersome and very expensive, and requires a guaranteed and continuous supply of LN
Another disadvantage is the risk of pathogen transmission between samples due to "dirty" LN or due to contaminated samples
Another disadvantage of storing biological samples in liquid nitrogen is the risk of tank failure and irreversible loss of samples
In addition to these inherent problems, the industrial production and distribution of LN and the energy requirements of specialized storage facilities have serious environmental impacts, leaving a large carbon footprint

Method used

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  • Device and method for freeze drying biological samples
  • Device and method for freeze drying biological samples
  • Device and method for freeze drying biological samples

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0150] sperm collection

[0151] Sperm samples were collected from n=3 rams of the Sarda breed and pooled for analysis as a single sample. Concentration and motility were assessed using CASA (Ivos, Hamilton Thorne, Biosciences). Only sperm exhibiting motility of 85% or higher were considered for this experiment. Sperm samples were diluted to a concentration of 50 million in Tris medium supplemented with LyoA solution containing 0.25M trehalose and 0.4M sorbitol or Lyo B solution containing 0.16M trehalose and 0.26M sorbitol and 20% egg yolk sperm / ml. Sperm were then cooled at 1°C / min to 4°C before motility was reassessed using CASA.

[0152] freezing

[0153] In this experiment, freezing was accomplished by pipetting 10 [mu]l of sperm droplets onto coverslips pre-cooled to various temperatures (-10, -25 or -35[deg.] C.) for 1 hour. The coverslip was then removed and warmed by placing it on a warm plate (38°C).

[0154] Freeze drying

[0155] We used a new device (ca...

Embodiment 2

[0193] Name: Freeze-dried human spermatozoa show high DNA integrity after UV irradiation compared with frozen spermatozoa.

[0194] Research question: DNA integrity of a) frozen human sperm compared to b) frozen / dried (lyophilized) human sperm after UV irradiation.

[0195] Short answer: Freeze-dried human sperm maintains high DNA integrity compared to frozen sperm.

[0196] What is known: It was recently shown that mouse sperm that had been stored in a desiccated state on a space station for 9 months and exposed to cosmic radiation showed only minor DNA damage, which was repaired by oocytes and produced normal offspring.

[0197] Study Design, Size, Duration: Human spermatozoa were collected and frozen and freeze-dried. DNA integrity was measured using Hallosperm for: 1. Fresh control, 2. Freeze dried and rehydrated, 3. Freeze dried irradiated and rehydrated, 4. Freeze irradiated and thawed.

[0198] Participants / Materials, Settings, Methods:

[0199]Fresh human sperm sa...

Embodiment 3

[0205] Embodiment 3 (use ovarian tissue)

[0206] Mouse ovaries were dissected and cut into 1×10×5 mm sections. Ovarian sections were exposed to Lyo solution containing 10% DMSO, 10% HAS in PBS. After exposure to the LYO solution, the sections were placed inside straws with special pods (also called capsules) as described in PCT WO / 2017 / 064715A1 and cooled at a rate of 1 °C / min using a Darya apparatus.

[0207] Figure 12 and 13A The drying step is shown in . Place the cells on the surface or in the vial on the shelf. The vacuum monitor indicated a pressure of 10 mTorr and the condenser was set at a temperature of -115°C. Drying at relatively high negative temperatures, ie primary drying, is carried out by keeping the shelf temperature slightly below the Tg' of -30°C. Secondary drying with Darya was done by raising the shelf temperature by 10°C every hour until the desired storage temperature was reached - which could be LN to RT. At the conclusion of the primary and / or...

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Abstract

A method and device for freeze-drying a biological sample of mammalian cells or tissue are disclosed. The method includes placing a biological sample on or in a structure to increase a temperature ofthe biological sample and with the biological sample in a closed chamber applying a vacuum to the chamber to lower a pressure within the chamber, cooling the chamber to lower a temperature within thechamber and applying heat to the biological sample within the chamber. The biological sample can include one or more of stem cells, hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, sperm, oocytes, embryos, ovarian tissue, uterine tissue or testicular tissue. A method for rehydration of a sample is further disclosed.

Description

[0001] Background of the invention [0002] This application claims priority to Provisional Application Serial No. 62 / 619,934, filed January 22, 2018, and Provisional Application Serial No. 62 / 634,868, filed February 25, 2018. The entire content of each of these applications is incorporated herein by reference. technical field [0003] The present application relates to methods of freeze-drying biological samples such as sperm, oocytes, embryos, reproductive tissues and stem cells and devices for performing such freeze-drying. Background technique [0004] Cryopreservation works very well for gametes of both sexes, as well as embryos of many domestic and wild species. Each species has its unique aspects, sensitivities and limitations, but germplasm can be cryopreserved, stored and ultimately used in assisted reproductive procedures. However, this effective method of cryopreservation is expensive. Storing cryopreserved samples under liquid nitrogen (LN) has high maintenan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0242A01N1/0257A01N1/0284A01N1/0289F26B5/06F26B21/10A01N1/0252A01N1/0294
Inventor A·阿拉夫
Owner 安全生殖公司
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