Kit for cell biosampling of aquatic organisms and hypothermic preservation of harvested samples, methods and uses thereof

Abstract

The present disclosure relates to a kit for cells biosampling and hypothermic preservation that enables the collection and preservation of viable cells from aquatic organisms, such as fish, molluscs, and crustaceans. The kit facilitates the hypothermic preservation of a variety of cell types, while minimizing trauma to the donor organism. Furthermore, it integrates components for maintaining cell viability during transport and long-term hypothermic storage.

Description

D E S C R I P T I O NKIT FOR CELL BIOSAMPLI NG OF AQUATIC ORGANISMS AND HYPOTHERMIC PRESERVATION OF HARVESTED SAMPLES, METHODS AND USES THEREOFTECHNICAL FIELD

[0001] The present disclosure relates to the field of biological sampling and hypothermic (2-8 °C), in particular on the collection of viable cells from aquatics organisms; namely fish, mollusc, crustacean, among others. The kit is designed for use in research, biotechnology, and cellular agriculture, where the preservation of cell viability is crucial for future applications, including for further cell line development, tissue engineering, and / or cellular agriculture production that require cellular integrity.BACKGROUND

[0002] As the field of cellular agriculture advances, there is an increasing demand for specific tools designed to collect biological samples that maintain high cell viability. Existing devices are not optimized for non-lethal biopsies of aquatic organisms.

[0003] The need for efficient biosampling tools for the collection and hypothermic preservation of viable cells is critical in the fields of cellular agriculture, marine biology, and biotechnology. Low temperature maintenance of cells is critical to preserve its integrity.

[0004] Current biosampling techniques for aquatic organisms, such as fish, molluscs, and crustaceans, are inadequate, often causing significant trauma or death to the donor organisms. The current methods and kits rely on biopsy techniques developed for human tissues and lack optimization for non-human species.

[0005] Moreover, there is no specific tool optimized for cell collection, preservation, and hypothermic preservation that addresses the specific needs of aquatic organisms.

[0006] There is the need to know exactly where and when the sampling was made in order to have a proper reference about the origin of the biological sample.

[0007] The information about the exact time of sampling is critical so that the further processing in the lab can consider this information.

[0008] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

[0009] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.GENERAL DESCRI PTION

[0010] The present disclosure relates to a kit for cells biosampling and hypothermic preservation that enables the collection and preservation of viable cells from aquatic organisms, such as fish, molluscs, and crustaceans. The kit facilitates the hypothermic preservation of a variety of cell types— such as gametes, blood, muscle, fat, and connective tissue - while minimizing trauma to the donor organism. Furthermore, it integrates components for maintaining cell viability during transport and long-term hypothermic storage.

[0011] An aspect of the present disclosure relates to a kit for collecting and preserving viable cells from an aquatic animal comprising: a biopsy-based sampling device for obtaining small aquatic animal / organism tissue sample with minimal trauma to the organism; pre-filled transport vials comprising a suitable growth and hypothermic preservation media for ensuring cell viability, that ensure cell viability during transport and until cell isolation.

[0012] In an embodiment, the kit for collecting and preserving viable cells from an aquatic animal may comprise: a biopsy-based sampling device for obtaining fish and molluscs tissue sample with minimal trauma to the organism; pre-filled transport vials comprising a suitable growth and hypothermic preservation media for ensuring cell viability, that ensure cell viability during transport and until cell isolation.

[0013] As used herein, the term "minimally invasive" refers to an intervention that accesses the target tissue with reduced trauma relative to an open surgical technique. Such an intervention typically involves the use of smaller incisions or natural access pathways and is characterized by decreased collateral tissue damage, reduced pain, diminished blood loss, and shortened recovery time, while providing diagnostic or therapeutic outcomes that are equivalent to or improved over those achieved with conventional open procedures.

[0014] As used herein, the term "reducing animal stress" refers to mitigating physiological and / or behavioral stress responses in aquatic animals associated with aquaculture practices, such as handling, grading, transport, stocking density, or water quality fluctuations. Such reduction is evidenced by improved physiological parameters (e.g., lowered cortisol, stabilized blood glucose, enhanced immune function), behavioral parameters (e.g., decreased erratic swimming, avoidance, or feed refusal), and overall welfare and performance outcomes (e.g., survival, growth, feed efficiency, or disease resistance).

[0015] In an embodiment, the kit further comprises a hypothermic preservation module that delays cell death, maintaining cell viability during the storage process until arrival to a laboratory for further processing. Biopsy tissue samples from fish and molluscs are maintained in a hypothermic preservation solution (e.g., PBS or DMEM or Leibovitz's L-15 + Heparin + Small molecules + Antibiotics + HEPES and FBS for improved tissue viability).

[0016] Ex vivo tissues from aquatic animals stored at 2-8 °C were shown to retain >70% cell viability for at least 24-48 hours, thereby supporting the feasibility of transport under the claimed conditions.

[0017] In an embodiment, the samples collected may be blood, gametes, muscle, fat, conjunctive tissue, and other viable cell types.

[0018] In an embodiment, the aquatic animal is a mammal, reptile, fish, mollusc, crustacean.

[0019] In an embodiment, the biopsy-based sampling device is capable of collecting multiple types of cells, including but not limited to gametes, muscle cells, blood cells, and / or connective tissue.

[0020] In an embodiment, the biopsy-based sampling device is capable of collecting multiple types of cells simultaneously, including but not limited to gametes, muscle cells, blood cells, and / or connective tissue.

[0021] In an embodiment, the kit of the present disclosure may further comprise a temperature-controlled containers with integrated data loggers for preserving samples during transport; preferably wherein the containers comply with UN3373 transportation protocols (https: / / www.un3373.com / enveloppeninfo / regulations) for biological materials.

[0022] In an embodiment, the kit of the present disclosure may further comprise a tagging system for sampled organisms, allowing for follow-up and data tracking.

[0023] In an embodiment, the kit of the present disclosure may further comprise an electronic system for recording and indexing sample metadata, including sample location, organism details, and / or storage conditions.

[0024] In an embodiment, the electronic system includes an automated SMS notification system, namely to alert laboratories when samples are being transported.

[0025] In an embodiment, the biopsy-based sampling device is a collection system for retrieving multiple tissue samples simultaneously.

[0026] In an embodiment, the hypothermic preservation solutions comprise a thermo gel pack, preferably a refrigerator gel pack (for example, sonoco thermosafe).

[0027] In an embodiment, the growth medium solution may comprise a basal media, specific for fish, molluscs, crustaceans and either other target organism; a specific serum that can be FBS or FBS equivalent; salts, as NaCI, KCI, MgSC , CaC or other; amino acids that can include L- glutamine, L-Lysine; vitamins as vitamin C, folic acid and complex B vitamins; energy sources as glucose, pyruvate or other; and pH buffers as NaHCOs, or HEPES; residual doses of antibiotics and / or antimycotic for potential contaminants as penicillin, streptomycin, amphotericin B or other.

[0028] In an embodiment, the kit of the present disclosure may further comprise a surgical patch procedure for minimizing aquatic animal stress.

[0029] Another aspect of the present disclosure relates to method for collecting and preserving viable cells, comprising the steps of: applying a biopsy-based sampling device to an aquatic organism to collect a small tissue sample; placing the tissue sample into a hypothermic preservation module designed to prevent cell death and preserve cell viability; the kit may further contain a physiological compatible medium to keep the samples in hypothermic temperature for the period of 18 and up to 24 hours, depending on how the samples are transported; transferring the preserved tissue into pre-filled transport vials for safe transport to a laboratory.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0031] Figure 1: Schematic representation of an embodiment of the box that transports the kit.

[0032] Figure 2: Schematic representation of an embodiment of the kit of the present disclosure wherein a) represents the gel packs; b) represents the vials pre-filed with preservation media; c) represents the needle for collecting samples; d) represents the mobile phone for recording the sampling and tagging the location and sending info to the lab.

[0033] Figure 3: Schematic representation of an embodiment of the biopsy needle device.DETAILED DESCRIPTION

[0034] The sampling kit of the present disclosure is able to collect and preserve viable cells from aquatic organisms, specifically targeting fish, molluscs, and crustaceans, while minimising the need for animal sacrifice whenever possible.

[0035] In an embodiment, the kit comprises a biopsy system, preferably a needle aspiration biopsy system to simultaneously collect multiple types of tissue, including blood, muscle, fat, gametes, and other biological materials, with minimal stress and no permanent harm to the organisms. Once collected, samples are stored in a protectant solution and transported in temperature-controlled containers that ensure cell viability. The kit also integrates digital systems for tracking, and monitoring samples, including automated notifications to laboratories for streamlined sample processing. Compliant with UN3373 transportation protocols, the kit of the present disclosure surprisingly addresses a critical need in cellular agriculture by providing an easy-to-use, ethical, and efficient tool for biosampling. Future versions of the kit will be adapted for use with birds, mammals, reptiles, and other organisms in cellular agriculture.

[0036] Figure 1 represents the box that transports the kit.

[0037] Figure 2 shows the components included in the kit of the present disclosure: a) the gel packs; b) the vials pre-filed with preservation media; c) the needle for collecting samples; d) the mobile phone for recording the sampling and tagging the location and sending info to the lab.

[0038] Figure 3 shows a detailed view of the biopsy needle device.

[0039] Table 1 shows a flow chart of the sample collection processCollection method

[0040] In an embodiment, minimally invasive biopsy protocols specifically adapted for aquatic species including fish, molluscs, and crustaceans, aiming to maximize cell viability while minimising animal trauma and sacrifice, particularly for fish and octopus, are described.Fish (e.g. Pagellus acarne, Gadus morhua)Step 1: Anesthetize the fish using MS-222 (tricaine) at 100-150 mg / L.Step 2: Disinfect the target site with chlorine based disinfectantStep 3: Using a 2-mm sterile biopsy punch, collect a skin / muscle sample from the dorsallateral region.Step 4: Apply gentle pressure to stop bleeding and surgical patch.Molluscs (e.g. Octopus vulgaris)Step 1: Collect the octopus carefully from the water and hold the animal securely but gently to avoid injury.Step 2: Choose one arm and identify the distal 0.5-1.0 cm segment to sample. Extend the arm and cut the tip segment.Step 3: Return the animal promptly to the water.

[0041] In an embodiment, the arm tip collection allows for epithelial and muscle sampling. This method is invasive and stressful; use only under ethical approvals.Preservation medium

[0042] In an embodiment, for the preservation and transport of tissue samples from octopus, fish, and other aquatic species, Leibovitz's L-15, DMEM, or PBS were used as base media. The base medium is supplemented with specific compounds to enhance sample stability and viability. Common additives include heparin (20 lU / mL) to prevent clotting, antibiotic- antimycotic solution (5-10%) to prevent microbial contamination, and HEPES buffer (10-20 mM) to maintain physiological pH. In some cases, 5%-10% foetal bovine serum (FBS) is added to improve cellular integrity during transport, and glucose (1 g / L) may be included to support minimal metabolic activity.Viability dataIn an embodiment, the protocol was successfully applied to both fish and octopus tissues, yielding viable cells (>70% cell viability) upon arrival at the laboratory following hypothermic transport.Packaging compliance

[0043] In an embodiment, all biological material shipments comply with UN3373 Category B standards for Biological Substance, Category B. The packaging system is composed of:Primary container:Sterile and sealed 15 or 50 mL Falcon tubes / vialsContain sample in preservation medium.Secondary container:Leak-proof, pressure-resistant plastic biohazard bag with absorbent material (to absorb all liquid if leakage occurs).Labelled with biohazard and UN3373 sticker.Tertiary container:Rigid outer box with at least 1.2 m drop-resistance.Includes thermal insulation + ice packs for hypothermic preservation.Labelled per IATA guidelines with sender / receiver info and UN3373 marking.

[0044] In an embodiment, the triple-containment system of the present disclosure meets the IATA Packing Instruction 650 and ADR 2023 transport regulations.

[0045] This invention was developed with financial support from "PRR Startup Portugal Vouchers with project n- 10171" research and innovation programme under grant agreement, leading to the present disclosure.

[0046] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

[0047] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples, including mixtures thereof.

[0048] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. "Detecting the presence of" can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0049] Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0050] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0051] The above-described embodiments are combinable.

[0052] The following claims further set out particular embodiments of the disclosure.

Claims

C L A I M S1. A kit for collecting and preserving viable cells from aquatic animals, comprising: a biopsy-based sampling device for obtaining small aquatic animal tissue sample; pre-filled transport vials comprising a suitable growth and hypothermic preservation media for ensuring cell viability; and a hypothermic preservation module.

2. The kit according to the previous claim, wherein the samples collected include blood, gametes, muscle, fat, conjunctive tissue, and other viable cell types.

3. The kit according to any of the previous claims, wherein the aquatic animal, reptile, fish, mollusc, crustacean.

4. The kit according to any of the previous claims, wherein the biopsy-based sampling device is capable of collecting multiple types of cells, including but not limited to gametes, muscle cells, blood cells, and / or connective tissue.

5. The kit according to any of the previous claims further comprising a temperature- controlled containers with integrated data loggers for preserving samples during transport; preferably wherein the containers comply with UN3373 transportation protocols for biological materials.

6. The kit according to any of the previous claims, wherein the transport package maintains the sample temperature between 2 °C and 10 °C for at least 24 hours under ambient conditions of 15-30 °C; preferably 2-8 °C; more preferably from 5-8 °C.

7. The kit according to any of the previous claims further comprising a tagging system for sampled organisms, allowing for follow-up and data tracking.

8. The kit according to any of the previous claims further comprising an electronic system for recording and indexing sample metadata, including sample location, organism details, and / or storage conditions.

9. The kit according to the previous claim, wherein the electronic system includes an automated SMS notification system to alert laboratories when samples are being transported.

10. The kit according to any of the previous claims wherein the biopsy-based sampling device is a collection system for retrieving multiple tissue samples simultaneously.

11. The kit according to any of the previous claims wherein the hypothermic preservation solution further comprises a suitable medium for support the in vitro culture of cells from aquatic species; said medium substantially mimic the physiological environment of marine or freshwater organism.

12. The kit according to any of the previous claims comprising wherein a thermo - gel pack, in particular a refrigerator pack, that allows to keep the biological samples in a hypothermic temperature range.

13. The kit according to any of the previous claims further comprises a surgical patch procedure for minimizing aquatic animal stress to be used whenever necessary.

14. The kit according to any of the previous claims wherein the hypothermic preservation media is a suitable medium composition, wherein said composition comprises 280 to 1050 mOsm / kg osmolarity, at least one biological buffers such as HEPES or sodium bicarbonate; a basal solution such as Leibovitz's L-15 medium, Dulbecco's Modified Eagle Medium (DMEM), or phosphate-buffered saline (PBS) supplemented with heparin, antibiotics, and optionally foetal bovine serum or glucose, among others.

15. A method for collecting and preserving viable cells from aquatic animal, comprising the steps of: applying a biopsy-based sampling device to an aquatic animal to collect a small tissue sample; placing the tissue sample into a hypothermic preservation module designed to prevent cell death and preserve cell viability; transferring the harvested tissue into pre-filled transport vials for safe transport to a laboratory.

16. The method for collecting and preserving viable cells from aquatic animal according to the previous claim comprising the following steps: applying a surgical patch to the biopsy site; immersing the sample in the hypothermic preservation medium; placing the preservation container inside the UN3373-compliant transport package; and monitoring and transmitting temperature data during transport via the digital tracking system.

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