WO2019156601A1

WO2019156601A1 - High efficiency cryo-additive agent to improve mammalian cells biopreservation

Info

Publication number: WO2019156601A1
Application number: PCT/SA2018/000006
Authority: WO
Inventors: Noha Abdullah S AL-OTAIBI; Nigel Slater; Hassan Rahmoune
Original assignee: Al Otaibi Noha Abdullah S
Priority date: 2018-02-08
Filing date: 2018-02-08
Publication date: 2019-08-15

Abstract

The present invention aims at enhancing the cryo -protective properties of media used for preserving mammalian cells by supplementing the media with Nigerose (Nig) as a cryo–additive agent. Using standard cryo -media (10% DMSO / 9% FCS) comprising Nigerose (Nig) reduces cellular damage by modulating oxido/redox pathways.Consequently, cell proliferation of cells cryopreserved in the presence of Nigerose is remarkable in compare to those processed in standard media.

Description

High efficiency cryo-additive agent to improve mammalian cells biopreservation

Technical field

The present invention introduces novel cryo-protective agents (CPAs) that provide cells with maximum protection during cryopreservation and boost cells recovery and proliferation post thawing.

Background

The present invention is focusing in the area of biopreservative media formulation to improve cryopreservation and maintenance of mammalian cells. Cryopreservation is the application of ultra- low temperature (-80°C up to - 196 °C [e. g. Liquid Nitrogen]) to cryopreserve cells and potentially tissues for a prolong period of time that extends between days to years. The application of the sub zero temperatures to cryopreserve cells in the presence of CPAs was found partially successful to halt the biological activities and maintain cells structure and functions intact by limiting cryodamage. The use of CPAs such as dimethylsulfoxide (DMSO), glycerol and trehalose showed a partial efficacy in maintaining cell integrity post thaw due to their cytotoxicity.

CPAs are commonly used in high concentrations to freeze living cells. Their primary protection mechanism is by interacting with and manipulating intracellular water during freeze-thaw. Flowever, CPAs commonly facilitates a partial recovery of less than 50% viable cells upon thawing. In addition, high concentration of CPAs may cause cytoxicity leading to cellular oxidative stress and causing cryo- injury. Consequently, the cryopreservation of mammalian cells in the presence of the classical CPAs (e. g. DMSO, Glycerol) precipitates the aging process of the cells and shortens their life span.

Over the last decade, a series of compounds were investigated for their properties to reduce cryodamage and enhance cell recovery post thaw. Flowever, these compounds¹-² seem to have limited or insignificant improve on cell status post-thawing. Flence, there is a need for effective cryo-protective additives to enhance the performance of standard cryomedia (e.g. glycerol, DMSO) with the overall aim to improve cell recovery and minimize cryo-damages.

Full description of the invention

Intense global industrial efforts are currently being spent on developing cell-based therapies for previously intractable diseases and some remarkable treatment outcomes, which have already been reported. For example, Novartis have recently obtained fast-track FDA approval for their cell-based CAR-T immunotherapy that has provided complete remission for a number of cancer patients. Such complex treatments are currently hampered by dependence on conventional cryoprotective media (CPAs) based cell cryopreservation protocols (e.g. Dimethyl sulfoxide (DMSO), glycerol and trehalose) as exposure to CPAs has been widely demonstrated to adversely impact cell quality. There are numerous reports of adverse patients' responses that are linked to exposing cells to DMSO prior subjecting patients to cell-based treatment. It is widely agreed in the biobanking/cryopreservation field that there is an urgent need for a novel cryopreservative agent to supplement existing DMSO or DMSO-free cryomedia. Here, we have found that pre-treating, freezing and thawing human nucleated cells in a formulation containing CPAs which includes DMSO plus Nig isolated from honey is able to dramatically reduce cryo-oxidation and maintain cell viability and function post freeze and thaw cycle (supporting claims data Fig 2 and 4). In particular, we have found that treatment of cells with Nig enables exceptionally good recovery of cell viability and function after freezing and thawing (supporting data Fig 3).

Furthermore, this natural product is extremely active at low concentration (300 mM) and derive from a natural source that have a long and widespread history of safe use as food products, with no known cytoxicity or adverse physiological behaviour in humans. This was reflected on our findings, where Nig promoted the activity of GR, an important enzyme in the oxido-redux pathway of glutathione, to (0.0013±0.00006 mU/ml), almost 2.4 times higher than in cells cryopreserved in DMSO alone. In addition, it significantly reduced LDH activity, a biomarker of stress level in cells, to (0.02±0.044 mU/ml), which is five times lower than DMSO alone (see supporting data Fig 4).

Moreover, Nig protected cellular proteins and lipids against oxidative damage (see supporting data Fig 5 and 6)

We have also established the biological and the molecular mechanisms underpinning Nig cryoprotective properties. We showed that treatment of cells with Nig prior to freezing extensively changes the proteome profile of the cryopreserved cells (e. g. reduction in the level of proteins associated with pro-oxidative status and increased cell metabolism and nuclear activities (supporting data Fig 2). Where appropriate the resulting proteomic profile associated with the cell

cryopreservation in the presence of Nig has been validated by functional assays (e. g. reduced LDH and increased GR activities as well as a reduction in protein carbonylation). We have further shown that cells treated with DMSO plus Nig, in comparison to DMSO alone, can be recovered to high viability post thaw, providing an excellent prospect of formulated cryopreservation media for cell based-therapy treatments (see supporting data Fig 3).

The further potential applications/ or use of declared features of the current discoveries claims the optimisation of the cryopreservation process by mixing a pre-determined quantity of one or more cryoprotectant molecules to be used in the cryopreserving/thawing media wherein: i) human/animal cells (including Stem cell, Infertility cells, islet cells) and/or human tissue (including embryonic tissues and organ transplants based therapies); ii) and/or Bio-preservation of biopharmaceuticals or food and crop protection industries.

References

1. Gibb, Z., Butler, T. J., Morris, L. H. A., Maxwell, W. M. C. & Grupen, C. G.

Quercetin improves the postthaw characteristics of cryopreserved sex-sorted and nonsorted stallion sperm. Theriogenology 79, 1001-1009 (2017).

2. Pallotta, V., Gevi, F., D’alessandro, A. & Zolla, L. Storing red blood cells with vitamin C and N-acetylcysteine prevents oxidative stress-related lesions: a metabolomics overview. Blood Transfus. 12, 376-87 (2014).

3. Kojibiose, O. F. Isolation of kojibiose, nigerose, maltose and isomaltose from honey.

Tohoku J. Agric. Res. 109-115 (1960).

Claims

The effective chemical entity in the cryomedia and declaring its properties for patent In this invention we claim that formulated cryomedia (DMSO/FCS, RPMI 1640) containing Nig is superior to conventional cryomedia (e. g. DMSO/FCS, RPMI 1640). Nig protects cells from cryo- oxidation and enhances cells recovery post thawing. The addition of Nig has the following effects:

1- Modulating cryo-damaged pathways by reducing the level of expression of the proteome associated with oxidation.

2- Increasing nuclear activities.

3- Enhancing reductive environment [e. g. reduced LDH and increased GR enzymatic activities and decreased proteins cabonylation] in cryopreserved cells resultant.

4- The modulation action of Nig is specific as it targets the reduction of protein

carbonylation/oxidation (supporting data Figure 6) rather than lipid oxidation only

(supporting data Figure 5).

Nigerose

It is a poly-phenol that naturally present in honey and black mold³. None of its cryo- properties were examined before. In our study, we have discovered for the first time that this molecule is able to protect cells during freeze-thaw cycle by reducing cryo-oxidative damage. We have also deciphered its mechanisms of action by establishing the proteomic and biological profiles underpinning Nig mode of action. The data showed in figures 1 & 2 showed that Nig promotes protein metabolism, energy pathways and oxidoreductase activities in cells post thaw on cryomedia containing Nig as a novel cryo-additive. 05

OH OH

Fig 1: Nigerose chemical structure. 3-O-a-D-glucopyranosyl-D-glucose

Invention elements to protect

1. The use/application of Nigerose as protective agent to improve cryo- and

maintaining media.

2. The antioxidant properties if Nigerose and its application as antioxidant

3. The modulation effect of Nigerose on cellular pathways

Biological samples and methods for evaluating cellular protection of the compound Human leukaemia cells (HL-60) were utilized for the present investigation. Cells survival rate, key functional enzymes and oxidative damaged macromolecules (lipids and proteins) were investigated prior and post preservation in conventional cryo-media DMSO with and without novel cryo-additive