Organotypical tissue slicer

Description

ORGANOTYPIC TISSUE SLICER Utility model field

[001] The present invention relates to the development of equipment for slicing biological tissues, such as brain, liver, and lung, into organotypic slices for experimental purposes. Organotypic slices are tissue preparations that maintain the three-dimensional structure and functionality of an organ or tissue in vitro, in well plates with culture medium, eliminating the need for animal experiments.

[002] Organotypic slices are used in scientific studies to investigate complex biological processes, such as disease development or response to treatments, in a controlled environment. However, to obtain them, it is necessary that the tissue be subjected to the slicing process, using the equipment proposed here, which becomes essential in the execution of this experimental model of organotypic slices. Fundamentals of the utility model

[003] Organotypic slices are experimental models that allow the study of tissue and organ biology in vitro, preserving their three-dimensional architecture and cellular interactions. This model is obtained by slicing tissues into thin layers, maintaining their physiological and functional characteristics, which allows for more precise and controlled investigations. The organotypic slice model arose from the need to develop methods that would allow the study of tissue physiology and pathology in a way that more closely resembles in vivo reality.

[004] The study of disease mechanisms is challenging due to the complexity of the interactions involved, requiring models Petition 870250070321, dated 11 / 08 / 2025, page 4 / 18 / 10 experimental models that resemble in vivo conditions. The main advantage of organotypic slices is their ability to more faithfully simulate the natural tissue environment, allowing the analysis of complex biological processes under conditions closer to reality. The organotypic slice model offers advantages over other experimental models, both in vitro and in vivo. For example, the use of primary or dissociated cell cultures does not reflect the complex cellular and molecular interactions of in vivo tissue. Conversely, although in vivo animal models are the closest to human physiological conditions, they have disadvantages such as high cost and time for disease induction.Furthermore, organotypic slices are more accessible and ethical from an animal welfare perspective than animal models, as they constitute an alternative for research requiring a more complex experimental system than conventional cell cultures, but without the need to use animals. To overcome these limitations without using in vivo models, organotypic slice cultures have been developed as a suitable alternative. This model attempts to replicate the natural conditions of the organism in vivo, maintaining the three-dimensional tissue cytoarchitecture and the interaction between different cell types, making these cultures a convenient model for investigating the behavior and physiology of a cell considering the cellular and environmental interactions of the original tissue.

[005] In the context of drug and treatment evaluation, organotypic slices offer an interesting experimental platform. Cultured slices can be exposed to different substances, such as drugs or therapeutic compounds, and the effects on cellular activity and tissue integrity can be monitored. This allows for the identification of new compounds with therapeutic potential and the evaluation of their efficacy and safety before testing in animal models or clinical trials. In addition, they are also useful for understanding the mechanisms of action of treatments applied to the slices.

[006] For biomedical research, the organotypic slice experimental model has been widely used to study neurodegenerative diseases, aiming to study adult neurogenesis in disorders of Petition 870250070321, dated 11 / 08 / 2025, page 5 / 18 / 10 Central nervous system (CNS) diseases such as Alzheimer's disease, Parkinson's disease, and cerebral ischemia, in order to investigate the effectiveness of new drugs and therapies, and to understand tissue development and regeneration processes.

[007] Furthermore, tissue slices such as cerebellum have also been used in studies of Multiple Sclerosis (MS), allowing the analysis of inflammatory and demyelinating processes associated with the disease. After establishing the slices in culture, drugs or molecules, or even demyelinating agents, such as lysolecithin, can be added to the medium to promote experimental demyelination or alter the rate of myelination or remyelination, allowing the screening of potential pro-remyelinating compounds. Therapies that affect remyelination in the CNS may be critical determinants of long-term functional outcome in MS, aiming not only at symptom relief but also at delaying disease progression.

[008] In addition to MS, which affects humans, in animals, Canine Distemper shares some similarities with MS in its pathophysiology. Both are autoimmune diseases, in which the immune system inappropriately attacks structures of the body itself. In MS, the immune system attacks components of the myelin sheath of neurons in the central nervous system, leading to demyelination and interfering with the transmission of nerve impulses. In canine distemper, the distemper virus infects cells of the central and peripheral nervous systems, resulting in an inflammatory response that can lead to demyelination and neurological dysfunction similar to that observed in MS. Thus, considering the similarities between the pathophysiology of MS and canine distemper, it is plausible to suggest that the organotypic slice model could also be beneficial in the study of distemper, contributing to the advancement of knowledge and the development of therapeutic strategies for both diseases.

[009] The method for obtaining organotypic slices involves a series of systematic and detailed steps to ensure the integrity and viability of the tissues. Initially, the organ or tissue of interest is removed from the research animal, and then the tissue is cut into thin slices, usually Petition 870250070321, dated 11 / 08 / 2025, page 6 / 18 / 10 with a thickness between 200 and 500 μm using a tissue slicer or vibratory microtome. The slices will then be cultured in well plates with culture medium containing 0.4 μm porous membrane inserts made of polytetrafluoroethylene (PTFE), which allow for an air-liquid interface, where the slices can be maintained for up to 4 weeks. The PTFE membrane inserts are permeable and inert, allowing molecules or therapeutic agents added to the medium to permeate the slice. The cultures can be monitored by observation under an inverted microscope, viewed through the bottom of the plate and the membrane insert.

[010] To obtain organotypic slices, specific equipment and techniques are used, such as the vibratory microtome and the McIlwain tissue slicer. The vibratory microtome allows for precise and uniform tissue cuts, preserving cellular integrity and three-dimensional architecture. Its operation is based on a vibratory blade system that allows for highly precise tissue cutting. First, the tissue is fixed in a special support, and then the microtome blade is positioned over the tissue. The blade vibrates rapidly in a specific direction, allowing the tissue to be cut into very thin slices. The slice thickness can be adjusted according to the needs of the experiment, ensuring uniform slices and preserving the integrity of cellular structures.

[011] The McIlwain tissue slicer is a classic piece of equipment used primarily for cutting soft tissues, such as brain and liver, into thin, uniform slices. Its operation involves the use of a sharp blade that is pressed against the tissue, allowing it to be cut into thin, uniform slices. The tissue is placed on a movable platform that can be adjusted to control the thickness of the cut. The McIlwain tissue slicer is especially useful for preparing tissue slices quickly and efficiently. Both pieces of equipment play an important role in the preparation of organotypic slices, ensuring the quality and reproducibility of the results obtained in experiments.

[012] However, although essential for the execution of the model Petition 870250070321, dated 11 / 08 / 2025, page 7 / 18 / 10 experimental organotypic slice analysis, this equipment is not always readily available in the routine of Brazilian researchers. This is mainly due to the lack of domestically manufactured products, such as the McIlwain Tissue Slicer, and scarce financial resources for research that would allow the purchase of these products from the international market. Furthermore, the high cost of this imported equipment represents a significant obstacle to national scientific development.

[013] Although there is already equipment on the market capable of cutting biological tissues into organotypic slices, called the McIlwain Tissue Slicer, there is a need for the development of national equipment that meets the researcher's needs and performs the same function. The main justification for this need is that the existing equipment is international and must be imported through a Brazilian company by Brazilian researchers who want to work with the experimental model of organotypic slices. Due to importation, the cost of the product increases significantly, since it is necessary to bear additional costs such as import tariffs, customs duties and expenses with international transport, in addition to currency fluctuations, considering that the price of the product is given in US dollars.

[014] According to a quote obtained on August 30, 2023 from the Brazilian company Leverfix, which imports the McIlwain Fabric Slicer, the total cost of the product, including shipping, was US$4,675.00, equivalent today to R$23,253.92.

[015] Due to the high cost and scarcity of resources for research in Brazil, acquiring a product like this becomes unfeasible for many researchers. This discourages them from working more with the organotypic slice experimental model as a replacement for in vivo animal models.

[016] The technical problem that motivated the development of the present utility model consists in the absence of a national equipment for Petition 870250070321, dated 11 / 08 / 2025, page 8 / 18 / 10 cutting of biological tissues capable of producing organotypic slices with precision and thickness control, combined with a significantly lower cost than imported models.

[017] The proposed model features a micrometric cutting system with linear and servomotors controlled by a digital interface, allowing automatic thickness adjustments and precise operation, while maintaining the quality of the organotypic slices obtained. This configuration reduces manufacturing and maintenance costs, eliminates the need for importation, expands access for Brazilian researchers to this tool and, consequently, contributes to the dissemination of the use of ex vivo models, in accordance with the 3Rs principles. Brief description of the drawings Figure 1 is an image of the external structure of the device, identifying the visible parts through a legend. Figure 2 is a schematic drawing of the device's components and their electrical connections that allow for its proper functioning. Description of the utility model

[018] The Organotypic Slice Cutter consists of a device composed of:

[019] 1 Switching Power Supply 10 A

[020] 1 TB6560 Motor Drive

[021] 1 Protoboard 200 points

[022] 1 Positive and Negative Line for 400-point Protoboard

[023] 1 Arduino Mega Atmega2560

[024] 1 12V Linear Motor with Sliding Nut Petition 870250070321, dated 11 / 08 / 2025, page 9 / 18 / 10

[025] 1 Servomotor 25kg

[026] 1 LCD Shield Display 16x2 with Keyboard for Arduino (Display Keypad)

[027] 2 Key Module End of Course

[028] 1 Linear Potentiometer 10KΩ

[029] 1 Knob Button for Potentiometer

[030] Jumper Cables

[031] Flexible Cables 0.50 mm

[032] 1 USB A / B Cable for Arduino

[033] 1 P4 Pigtail Cable for Power Supplies

[034] 1 Power Cord

[035] 1 Rocker Key

[036] 1 Steel Blade for Fine Cutting (razor blade)

[037] External structure and attached components of the motors made by 3D printing with PLA (Polylactic Acid).

[038]

[039] After connecting the device to the power outlet (110 or 220 V) and turning on the rocker switch, the display lights up showing the message Let's slice?, and below it Menu. The Menu has 4 options: Start, Advance and S0, each with its specific functionality which will be detailed below.

[040] First, after selecting the Start function, a submenu opens with different cutting size options: 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, and 500 μm. After selecting the chosen cutting size, the linear motor and servo motor start operating as follows: the linear motor carries a 3D printing structure that serves as a table to support the fabric to be sliced. When the linear motor gives a Petition 870250070321, dated 11 / 08 / 2025, page 10 / 18 / 10 step, it moves the selected size in micrometers, also moving the table with the fabric. The servomotor, in turn, carries a 3D printing rod that allows the attachment of a steel blade for fine cutting (razor blade). After the linear motor moves one step with the selected size, the servomotor lowers the rod with the blade at a 90° angle, cutting the fabric on the table. During the operation of the motors, it is possible to adjust the cutting speed by regulating the potentiometer. This adjustment directly controls the operating speed of the servomotor, influencing the lowering movement of the rod attached to it and, consequently, the rate at which the cut is made.

[041] The motors continuously perform this operation, cutting the entire tissue sample, until the linear motor reaches an end of stroke, which causes the motors to stop.

[042] The Advance function is responsible for moving the linear motor forward, shifting the table where the sample is positioned. After being selected in the menu, it directs the linear motor to advance forward, allowing the sample's positioning to be adjusted. This functionality is essential for positioning the beginning of the sample under the cutting blade, ensuring that the cutting process can be started accurately.

[043] The S0 function, in turn, is the initial position function (in physics, the expression S0 is used to refer to the initial position of a body). Thus, after being selected in the Menu, it brings the linear motor with the cutting table back to the position where it started cutting, stopping after activating a limit switch that marks its initial position. After returning the linear motor to the initial position, the motors are ready to start a new slicing operation.

[044] Finally, there is also the Reset function, activated by a button on the keypad display, which is intended to stop the operation of the motors after the Start function has been activated. This function allows the slicing process to be interrupted before completion, if necessary for any reason.

[045] It is noteworthy that the different cut sizes were calibrated for Petition 870250070321, dated 11 / 08 / 2025, page 11 / 18 / 10 to ensure its accuracy. To this end, the table under the linear motor was covered with modeling clay, allowing the blade, when activated by the servomotor, to leave markings on the surface. Then, the distance between these markings was measured with a digital caliper, verifying if the cutting interval corresponded to the desired measurements.

[046] The diagram of the external structure, detailing the motors and their attachments, as well as the internal part, detailing the electrical connections, are presented in Figures 1 and 2, respectively. Examples of implementations of the utility model

[047] In the present invention, the financial inconveniences of existing equipment on the market are avoided, since it would be produced nationally, not requiring importation, and would be more economically viable for acquisition by Brazilian researchers. Thus, the creation of a national device, more economically viable for Brazilian researchers, would help to further disseminate the organotypic slice experimental model. This model is important because it allows the study of important diseases, such as neurodegenerative diseases, which are increasingly prevalent in society.

[048] Furthermore, the model is aligned with the 3 R's Principle in animal experimentation (Reduction, Refinement, Replacement), as it allows for a reduction in the use of animals in research. With just one mouse brain, it is possible to obtain several organotypic slices, which can be used as multiple triplicates in experiments, maximizing the use of a single animal. In addition to reducing the number of animals used, this experimental model also minimizes the performance of experiments that could cause suffering to the animals. This is because the studies are conducted using animal tissue cultivated in vitro after euthanasia, eliminating the need for experiments performed in vivo.

[049] The pre-existing equipment has a classic structure, Petition 870250070321, dated 11 / 08 / 2025, page 12 / 18 / 10 predominantly manual and analog, reflecting the fact that the organotypic slice experimental model has been studied for many years. For this reason, the available equipment is technologically outdated, highlighting the need for modernization. This update is essential so that this important experimental model can be widely explored and accessible to researchers in Brazil and worldwide.

[050] In this context, the proposed invention offers significant advantages over existing equipment, providing improvements that directly contribute to the modernization and efficiency of the device. The invention presented here proposes a digital design, with a display that presents a menu of options that control the equipment. Unlike the proposed invention, the pre-existing equipment does not have a display or digital control, presenting a more rudimentary structure. In the developed solution, the control and integration of functions are performed by means of an Arduino Mega, programmed to manage the operation in an automated and efficient manner, providing greater precision and practicality during use.

[051] The invention is simple, functional and intuitive to use. However, although simple in its functionality, it is efficient in fulfilling the proposed objective of cutting biological tissue into organotypic slices of micrometric thickness.

[052] Finally, it is declared that the present invention is of original design, since no pre-existing product manual was used as a basis for the planning and construction of the equipment. Thus, the mechanical and electrical structure was developed for the equipment in a completely original manner, resulting from study and research to define the most appropriate materials and how to integrate them within an electrical circuit. Petition 870250070321, dated 11 / 08 / 2025, page 13 / 18

Claims

1. ORGANOTYPIC TISSUE SLICER, characterized by comprising a cutting structure equipped with a linear motor configured to move a tissue support table in a micrometric manner and a servomotor coupled to a steel blade to perform precise cuts, the operation being coordinated by a digital control system based on a programmable microcontroller that allows selecting different cutting thicknesses through an interface with a display and interactive menu.

2. ORGANOTYPIC TISSUE SLICER, according to claim 1, characterized by allowing the selection of cutting thicknesses between 250 μm, 300 μm, 350 μm, 400 μm, 450 μm and 500 μm.

3. ORGANOTYPIC TISSUE SLICER, according to claim 1, characterized by the linear motor moving the tissue support table in controlled displacements and the servomotor actuating the blade to perform the cut immediately after each movement of the linear motor.

4. ORGANOTYPIC TISSUE SLICER, according to claim 1, characterized by having control functions that include “Start” to start the operation of the motors, “Advance” to move the linear motor forward, “S0” to return the linear motor to the initial cutting position and “Reset” to stop the process before completion.

5. ORGANOTYPIC TISSUE SLICER, according to claim 1, characterized by enabling the obtaining of multiple organotypic slices from the same sample, contributing to the reduction of the use of animals in scientific experimentation and aligning with the principles of the 3 Rs (Reduction, Refinement, Replacement).

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