Apr 10, 2025

Public workspaceExplant in natura and cryopreserved myometrial tissue for in vitro cell culture of Primary Smooth Muscle from Myometrium of Ovis Aries (PSMo24)

DOI
https://dx.doi.org/10.17504/protocols.io.q26g75ze3lwz/v1
  • Carolina Rodriguez Jimenez1,
  • Bruno Scatena Gatti1,
  • Livia Maria Presuto1,
  • Patricia Spoto Corrêa1,
  • Severino Matias de Alencar2,
  • Helder Louvandini1
  • 1Laboratory of Animal Nutrition, Center for Nuclear Energy in Agriculture, University of Sao Paulo, 303 Centenario Avenue, Piracicaba, SP 13416-000, Brazil;
  • 2Cell Cultivation Laboratory, Department of Agroindustry, Food and Nutrition, School of Agriculture "Luiz de Queiroz" (ESALQ) University of São Paulo (USP), Piracicaba, Brazil
Carolina Rodriguez Jimenez
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g75ze3lwz/v1
Protocol Citation: Carolina Rodriguez Jimenez, Bruno Scatena Gatti, Livia Maria Presuto, Patricia Spoto Corrêa, Severino Matias de Alencar, Helder Louvandini 2025. Explant in natura and cryopreserved myometrial tissue for in vitro cell culture of Primary Smooth Muscle from Myometrium of Ovis Aries (PSMo24). protocols.io https://dx.doi.org/10.17504/protocols.io.q26g75ze3lwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 24, 2025
Last Modified: April 10, 2025
Protocol Integer ID: 124907
Keywords: uterus, muscle, reproduction, sheep, culture, cell culture of primary smooth muscle, primary smooth muscle from myometrium cell, primary smooth muscle from myometrium, studying primary smooth muscle, tissue culture, specific muscle cell, immediate acquisition of uterine tissue, myometrium cell, muscle cell, myometrial tissue, psmo24 cell culture, myometrium of ovis ary, primary smooth muscle, uterine tissue samples from ovis ary, cell culture, uterine tissue sample, establishment of psmo24 cell culture, uterine tissue, fragments of myometrial tissue, myometrium, subsequent cell explantation, standard in cell biology study, tissue sample, cell biology, cell biology study, fresh cell, similar to the in vivo state, cell passage, cell proliferation, physiological mechanisms with other cell type, muscle, uterus, protocol for cryopreservation, comprehensive understanding of cellular function, human biotechnology, vivo state, tissue, cell
Funders Acknowledgements:
CNPq
Grant ID: 403541/2021-2
FAPESP
Grant ID: 2019/26042-8
Disclaimer
The information presented in this article aims to provide general knowledge on the topic discussed. The use of this information is the sole responsibility of the reader. The author is not responsible for any direct, indirect, or consequential damages resulting from the use or interpretation of the data presented here.
Abstract
Since the first in vitro cell culture was conducted in 1907 by the American Ross Granville Harrison to the present day, cell culture has established itself as an essential tool for basic and applied research, covering areas such as cell biology, molecular biology, physiology, animal and human biotechnology, among others. Cell culture is widely recognized as the standard in cell biology studies due to its ability to mimic the cellular environment in a way similar to the in vivo state. In the context of studying primary smooth muscle from myometrium cell, beyond their reproductive importance, these cells share physiological mechanisms with other cell types, allowing for a more comprehensive understanding of cellular function and specific muscle cells. One of the main challenges in establishing primary culture is the immediate acquisition of uterine tissue. To overcome this limitation, this study describes the development of a methodology for primary smooth muscle from myometrium of Ovis Aries (PSMo24) and establish a protocol for cryopreservation of myometrial tissue followed by subsequent cell explantation. This approach aims to maximize the uterus’ biological potential and minimizing tissue waste, which is traditionally used only in fresh form. Moreover, given the restrictions on animal’s experimentation, tissue culture stands out as an alternative method, providing valuable knowledge to evaluate substances. This contributes to the reduction or replacement of animal experimentation, aligning with ethical and scientific principles in biological studies. For the establishment of PSMo24 cell culture, uterine tissue samples from Ovis Aries were collected and subjected to enzymatic digestion. Simultaneously, fragments of myometrial tissue were cryopreserved, thawed, and explanted for cell culture. Ten cell passages were performed, along with quantification, morphology analysis, cytotoxicity assessment, antioxidant capacity evaluation, and RNA extraction. Here, we validated the protocol for PSMo24 from fresh cells and cryopreserved tissue for explant by demonstrating the morphology, viability, and cell proliferation, indicating new possibilities for their aplication in studies using reproductive and muscle cells, as well as in pharmacological or bioproducts tests in animals and humans.
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Guidelines
The propose a methodology for the primary culture of smooth muscle cells from the myometrium of Ovis aries (PSMo24), emphasizing the use of both fresh and cryopreserved tissue. The protocol includes uterine tissue collection, enzymatic digestion, cell explantation, cell passaging, and morphological, functional, and molecular analyses. The aim is to optimize the use of uterine tissue, reduce the use of animals in research, and expand the applications of these cells in reproductive, muscle, and pharmacological studies.
Materials
ReagentSilica ResinCarolina Biological SupplyCatalog #C33426
ReagentsCompanyCode
ABAP (Ácido 2,2'-azino-bis(3-etilbenzotiazolina-6-sulfônico), solução a 1 mM)Sigma-Aldrich440914
Amphotericin B SolutionSigma-AldrichA2942
Antibiotic/AntimycoticSigma-AldrichA5955
Collagenase from Clostridium histolyticumSigma-AldrichC9891
DAPI - 4′,6-Diamidine-2′-phenylindole dihydrochlorideSigma-AldrichD9542
DCFH-DA  2',7'- Dichlorofluorescin diacetateSigma-AldrichD6883
Dimethyl sulfoxide - DMSOSigma-AldrichD2650 
Dulbecco’s Modified Eagle’s Medium/High ModifiedSigma-Aldrich56436C
Ethylene glycol Sigma-Aldrich324558
Fetal Bovine SerumSigma-AldrichF7524
Formaldehyde solutionSigma-Aldrich818.708
GlycerolSigma-AldrichG7757
Ham' Nutrient Mixture F12Sigma-Aldrich51651C
Hanks’ Balanced Salt solutionSigma-AldrichH6648
HEPESSigma-AldrichH6147
MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)Sigma-AldrichCT01-5
penicilina/estreptomicinaSigma-AldrichP4333 
Phalloidin 488 Sigma-Aldrich49409
Phosphate Buffered SalineSigma-Aldrich806544
Quercetin Solution Sigma-AldrichQ4951
SacaroseSigma-Aldrich50389
Sodium BicarbonateSigma-AldrichS5761
Sodium chlorideSigma-AldrichS5886
Solução de azul de tripano a 0,4%.Sigma-AldrichT6146
Triton X-100 Sigma-AldrichT8787
Trypsin-EDTAGibcoT2610
Absolute EthanolÊxodo CientíficaAG07145RA
Isopropyl alcoholÊxodo CientíficaAI09965RA
Tri ReagentSigma-AldrichBCCB0163
ChloroformÊxodo CientíficaC09877RA
Reagents for Primary Smooth Muscle Cell Culture from Ovis aries Myometrium

Material For Cell Culture (Sterile)
Vacuum Filter System for Bottles
Syringes (10 mL and 20 mL)
Sterile Pipettes
Sterile Filter (if necessary)
Sterile Falcon Tubes (15 mL and 50 mL)
Sterile 0.22 µm Filter
Shoe Covers
Scalpel Handle
Scalpel Blade
Petri Dish
Parafilm
Paper Towel
Mask
Glass Tip
Glass Rod
Glass Bottles – Blue Cap
Falcon Tube Racks
Disposable Gown
Disposable Gloves
Cell Culture Bottles
Bottle with Alcohol
Beaker
Aluminum Foil
1-Liter Volumetric Flask
Material for cell Culture to Primary Smooth Muscle Cell Culture from Ovis aries Myometrium
EquipmentCompany
Inverted MicroscopeBel photonics
Heating PlateTed
Flow 1 (Biological Safety Cabinet) Pachane pa400eco
Flow 2 (Biological Safety Cabinet)Pachane pa70
Refrigerator Consul frostfree
FreezerColdlab 
Incubator 1Binder
pH Meter 2 (Fixed)Hanna edge pH
Water BathBenchmark  scientitic   mybath 8l
BalanceBel engineering
Magnetic StirrerFisotom
Ultrasonic BathSolidsteet
Vortex MixerNova
Equipment for cell Culture to Primary Smooth Muscle Cell Culture from Ovis aries Myometrium


Troubleshooting
Safety warnings
This protocol does not involve hazardous procedures; however, it is essential to follow standard biosafety practices to protect both the researcher and the cellular material. The use of appropriate personal protective equipment (PPE). Additionally, all procedures should be conducted under aseptic conditions in a cell culture laboratory to prevent contamination and ensure the integrity of the biological material.
Ethics statement
The following protocol was submitted to the ethics committee on animal use of the Center for Nuclear Energy in Agriculture from University of São Paulo (CENA/USP/Protocol No. 0005/2022).
METHOD I - EXPLANT OF FRESH MYOMETRIAL TISSUE FOR THE IN VITRO CULTURE OF PSMo24 CELLS: PRIMARY SMOOTH MUSCLE FROM THE MYOMETRIUM OF OVIS ARIES
3w 3d 1h 25m
The myometrial tissue was collected from five non-pregnant Santa Inês ewes, aged between 1 and 2 years, in perfect health condition.
Uterus collection: At the slaughterhouse, use your personal protective equipment and avoid all contaminants in the area. Carefully remove the uterus from the ewe using a sterile blade and place it in a sterile Petri dish.
First wash: Immediately after collection, wash the uterus with 70 % alcohol (±Temperature5 °C ) for Duration00:00:30 to sterilize the external surface and reduce the presence of contaminants.
Second wash: Wash with tissue washing medium Go to 0.9 % Sodium Chloride supplemented with penicillin and streptomycin antibiotics (pen+strep) at ±Temperature5 °C .

Cutting of sample I: In a previously aseptically prepared area at the slaughter site, make an initial large cut of approximately Thikness18 cm ^2 of tissue from each uterine horn, as shown in Figure 1.

Figure 1: Process of obtaining and preparing ovine myometrial tissue for in vitro culture. A) Macroscopic visualization of the ovine uterus after dissection; B) Fragmentation of myometrial tissue into explants (indicated by arrows and dotted circles); C) Myometrial explants placed in culture flasks containing specific medium.


Transport solution: Place the sample in aAmount50 mL Falcon tube with transport solution, 0.9 % Sodium Chloride supplemented with antifungal atTemperature5 °C Go to . Keep the sample for no more than Duration02:00:00 under these conditions to ensure tissue integrity before proceeding with the next steps of the protocol.

Transport: Transport the samples into a cooler at Temperature5 °C to the laboratory.
Safety information
Do not expose the sample to extreme temperatures or sudden temperature changes during transport.

Tissue fragmentation: In the cell culture laboratory, sanitize the Falcon tubes and bring them inside the laminar flow hood. Transfer the sample from the Falcon tube to a sterile Petri dish with Phosphate Buffered Saline (PBS) and antibiotics (cell washing medium Go to ). Cut the tissue into smaller fragments of approximately Thikness0.5 cm ^2 , avoiding the collection of the caruncle area (Figure 1 Go to ).

Washing of the fragments: Add several aliquots of Amount200 µL of antibiotics to a Petri dish. Briefly immerse each fragment for initial disinfection, then transfer the fragments to a new cell washing mediumGo to .
Note
This step is essential to ensure the removal of potential impurities or contaminants.

Enzymatic digestion: Place four fragments in Amount15 mL Falcon tubes containing aAmount1 mg /mL collagenase solution. Place the tubes in an incubator at Temperature37 °C with 5 % CO₂ and controlled humidity until shaking occurs.

a. Periodic shaking: Manually mix the solution every Duration00:10:00 for a total period of Duration00:30:00 , up to a maximum ofDuration00:40:00 . Shaking helps the collagenase digest the tissue so that the cells can be released for subsequent culture.
b. Visual observation: Interrupt the digestion as soon as the solution turns white or milky. Avoid allowing the tissue to become viscous or "slimy," which indicates excessive digestion.
c. Centrifugation: After digestion, centrifuge the tubes at Centrifigation1194 x g, 00:05:00 to separate the cells from the collagenase. Discard the supernatant and wash the fragments with PBS to eliminate enzyme residues.

1h 25m
Distribution into culture bottle: Transfer the fragments to a 25 cm² culture bottle and add Amount4 mL of culture medium (Dulbecco´s Modified Eagle´s Medium/High Modified; Ham´s Nutrient Mixture F12; Fetal Bovine Serum; and antibiotics). The medium should cover the fragments shallowly to optimize cell adhesion. Place the bottle in the incubator Temperature37 °C , 5 % CO₂. Keep the culture undisturbed for Duration96:00:00 to allow the cells to begin adhering and proliferating on the fragments. On the fourth day, check the confluence and add Amount2 mL of culture medium Go to .

4d
Incubation
Regular medium change: Starting on the fifth day, perform medium changes and continue every three days to renew nutrients.

a. Washing: Using a glass pipette adapted to a vacuum pump, remove the culture medium and add Amount3 mL of PBS with antibiotics (cell washing medium Go to ).
b. Medium replacement: Add Amount3-4 mL of culture medium Go to as the cells detach from the myometrial tissue.


Removal of tissue fragments: Between day 7 and day 10 of culture, remove the tissue fragments to prevent contamination and promote cell expansion. By day 10, cellular cluster should be present.
Maximum passage duration: Maintain the culture of the first passage for no more than 20 days. If confluence is not reached by this period, it is not recommended to proceed. In Figure 2, you can observe the sequence of PSMo24 in natura until their confluence (80 %).

Figure 2. Morphology of Primary smooth muscle from the myometrium of Ovis Aries (PSMo24) cells, obtained from fresh tissue, at different stages of in vitro culture. (a) Cells after initial adhesion to the substrate; (b) Initial cell proliferation; (c) Partial monolayer formation; (d) Monolayer at higher confluence; (e) Cells at an advanced growth stage; (f) Cells at 80 % confluence.

2w 6d
Critical

Note
The ruminant uterus caruncles are a dense connective tissue that is highly vascularized, covered by a simple cuboidal or low columnar epithelium. They are located in the uterine mucosa with attachment sites for the cotyledons of the fetal placenta. Therefore, they are not smooth muscle tissue and should not be collected or cultured in this protocol.

Note
Perform at least 4 culture bottles per uterus and/or animal to ensure the growth and storage of future cells.

METHOD II - DETACHMENT OF PSMo24 CELLS-TRYPSINIZATION
1d 0h 14m
Once approximately 80 % confluence is reached, remove the culture medium and add Amount3 mL of cell washing medium Go to .

Addition of Trypsin-EDTA: Add the trypsinization medium Go to : PBS + trypsin to the 25 cm² culture bottle.

Incubation: Place the bottle in the incubator (Temperature37 °C ; 5 % CO₂) forDuration00:08:00 . Observe under an inverted microscope to check if the cells have detached. If there are still adherent cells, incubate for up to a maximum of Duration00:14:00 .
8m
Incubation
Imaging
Trypsin inactivation: Add Amount3 mL of culture medium Go to to neutralize the trypsin as soon as the cells are completely detached.

Perform the counting: Count the cells using a Neubauer chamber or an automatic cell counter, ensuring an adequate cell quantity for culture and storage.
Analyze
Counting and viability of Primary Smooth Muscle Cells from Myometrium in the Neubauer Chamber

After trypsinization of the cells or thawing of the cells, transfer the cell suspension to a sterile Falcon tube. Collect Amount10 µL of cell suspension + Amount10 µL of 0.4 % trypan blue. Gently mix and let it react for Duration00:01:00 at TemperatureRoom temperature .
1m
Clean the Neubauer chamber and coverslip and allow them to dry. Add Amount10 µL to the chamber, allowing the liquid to fill it by capillary action.
Count the live (unstained) and dead (stained blue) cells in at least 4 large quadrants and calculate the viability percentage. For quantification, use the following equation:

Note
Cell Counting = NCC/NQ * Vol * 104 * 2

Where:
  • NCC: Total Number of Cells Counted
  • NQ: Number of Quadrants Counted
  • Vol: Volume of Cell Suspension Collected (mL)
  • 104: The correction factor adjusts the calculation to the scale of the volume analyzed in the Neubauer chamber.
  • When viability is not performed, remove the dilution factor (2).


Centrifuge and Separate: After counting, distribute the appropriate number of cells for reculturing or cryopreservation. Transfer the contents to Falcon tubes and centrifuge at Centrifigation428 x g, 00:05:00 , then discard the supernatant.


Note
Small flask (P): 25 cm² – 500,000 cells

Medium flask (M): 75 cm² – 1,500,000 cells

Large flask (G): 182 cm² – 3,640,000 cells


5m
Centrifigation
Part 1 (Criopreservation): Add Amount1 mL of the cryopreservation solution Go to (Dimehylsulfoxide - DMSO + Fetal Bovine Serum - FBS), place in a cryogenic tube, and place in Mr. Frosty, a cryogenic container that provides a gradual cooling rate of Temperature1 °C /min. After Duration24:00:00 , transfer to the nitrogen tank (Temperature-196 °C ) for indefinite storage.

1d
Pause
Part 2 (Continuity of Culture): Add the acquired cell quantity in Amount5 mL , Amount15 mL , or Amount25 mL of culture medium to a 25, 75, or 182 cm² culture flask, respectively. Return to the incubator for culture until a new confluence is reached.

Incubation
METHOD III - SUBCULTURE AND THAWING OF PSMo24 CELLS
6m
Subculture of PSMo24 Cell Passages

After performing the explant and cryopreservation of passage 1, continue with the subsequent passages (maximum of 10). Keep subculturing and changing the culture medium every 3 days, maintaining the culture until the desired cell confluence is reached, which usually occurs between 3 and 9 days. Once confluence is satisfactory, repeat Method II - Trypsinization.
From this point, the necessary analyses or experimental tests can be performed directly on the cells. To ensure the continuity of the experiment in case of unexpected issues with the cultures, it is recommended to regularly freeze cell passages to enable future subcultures.
Thawing of cryopreserved PSMo24 cells for subculture
Remove the cryotube from the liquid nitrogen tank, keep it at TemperatureRoom temperature for Duration00:01:00 and then complete the thawing process at Temperature37 °C in a water bath.
1m
In the laminar flow hood, transfer the entire content of the cryotube (Amount1 mL ) into a Falcon tube containing Amount4 mL of culture medium Go to .

Collect Amount10 µL of the suspension to perform the cell count (item) and determine the appropriate culture flask.
Centrifuge for Centrifigation428 x g, 00:05:00 and remove the supernatant without disturbing the cell pellet.
5m
Add culture medium Go to to the tube containing the cell pellet and resuspend, homogenizing with back-and-forth movements.

Place the suspension in the chosen culture flask and complete the medium if needed. Incubate in the incubator with 5 % CO2 at Temperature37 °C .
METHOD IV - EXPLANTATION OF FROZEN MYOMETRIAL TISSUE FOR IN VITRO CULTURE OF PSMo24 CELLS
23m
Collection and Preparation of Myometrial Tissue, see item Go to to Go to .
Freezing Process (vitrification or rapid freezing)
With the tissue fragments prepared, add Amount2 mL of cryopreservation medium (Ethylene glycol - EG, Sucrose - SAC, FBS, and Dulbecco´s Modified Eagle´s Medium - DMEM Go to ) to each well of a 24-well plate. Then, immerse two fragments in each well, keeping them submerged for Duration00:05:00 After the exposure period, transfer four fragments to a cryotube containing Amount1 mL of cryopreservation medium Go to and immediately store in liquid nitrogen atTemperature-196 °C .
5m
Thawing of Myometrial Tissue
Remove the cryotube from the liquid nitrogen and leave it at TemperatureRoom temperature for about Duration00:01:00 . Then, immediately transfer it to a Temperature37 °C water bath and maintain it until fully thawed, which usually takes Duration00:02:00 . Prepare the sequential washes to be performed with DMEM and Sucrose (0.25 and 0.5 M), referred to as thawing I,II, e III (DMEM-Pure). After preparing the solutions, carry out the sequential washes as indicated:

3m
Wash I:
Place two tissue fragments in each well with Amount2 mL of thawing solution I Go to and keep them for Duration00:05:00 . After this period, transfer the fragments to thawing solution II Go to .
5m
Wash II:
Keep the fragments immersed in thawing solution IIGo to for Duration00:05:00 . Then, carefully transfer the fragments to thawing solution III (DMEM-Pure).

5m
Wash III
Keep the fragments in thawing solution III (DMEM-Pure) forDuration00:05:00 After this time, wash the fragments with cell washing medium Go to and proceed with the enzymatic digestion, as mentioned in the item Go to . In Figure 3, we can observe the detachment of cells after the explantation of cryopreserved tissue until it reaches 80 % confluence.

Figure 3: Morphology of Primary smooth muscle from the myometrium of Ovis Aries (PSMo24) cell, obtained from cryopreserved tissue, at different stages of in vitro culture. (a) Cells emerging from the tissue and proliferating; (b) Initial cell proliferation; (c) Distribution in the culture flask; (d) Cells at 80 % confluence.

Note
After the fragments come out of the water bath, everything that comes into contact with the tissue must be at Temperature37 °C .

5m
METHOD V - MORPHOLOGICAL VALIDATION BY FLUORESCENCE CONFOCAL MICROSCOPY WITH DAPI AND PHALLOIDIN STAINING OF PSMo24 CELLS
3d 5h 50m
Culture the PSMo24 cells as described in itemsGo to ,Go to and Go to . After confluence, perform trypsinization as described inGo to

I a 96-well plate, resuspend the PSMo24 cells at a concentration of 2.0 × 10⁴ cells/well in Amount200 µL of culture medium Go to . Incubate the plate atTemperature37 °C in a CO2 incubator for Duration24:00:00 .

1d
After 24 hours, observe the cell adhesion under the inverted microscope; Remove the culture medium by carefully inverting the plate, Wash the cells once with pre-warmed PBS at Temperature37 °C , Ph7.4 .

Apply Amount100 µL of the treatments of interest, diluted in serum-free culture medium (Go to )  in quadruplicate in the 96-well plate.

Incubate the plate at Temperature37 °C in the CO2 incubator for Duration04:00:00 or for the necessary duration of the treatment reaction. After the incubation time, remove the treatments by carefully inverting the plate.
4h
Fix the sample in 3.7 % formaldehyde solution without methanol Go to in PBS forDuration00:20:00 atTemperatureRoom temperature .

20m
Wash once with PBS.
Then, permeabilize with 0,3 % Triton X-100 in 1 % Bovine Serum Albumin (BSA) Go to for Duration00:30:00

30m
Incubate the cells in a dilution of: 1:10,000 of 4′,6-Diamidine-2´-phenylindole dihydrochloride (DAPI) Go to and 1:400 of 1:400 of phalloidin 488 Go to , prepared in 1 % Triton X-100 in 1 % BSA Go to .

Add Amount100 µL of a solution containing DAPI and Phalloidin 488 Go to to each well of the 96-well plate. Incubate the plate at TemperatureRoom temperature for Duration01:00:00 , keeping it in the dark to protect the fluorophores.

1h
Wash once with PBS.

Add 1 drop per well of the antifade solution Go to and analyze within Duration48:00:00

Note
To remove the reagents from the 96-well plate, carefully invert the plate completely.

2d
Set the inverted confocal microscope to the excitation/emission channels of DAPI (358–461 nm, blue) and Phalloidin 488 (488 nm, green), configuring the acquisition parameters (laser intensity, gain, and resolution) according to the sample. During analysis, identify the green fluorescence to visualize actin filaments in the cytoplasm and the blue fluorescence to examine the integrity of the nuclei. Capture images that shows the cellular organization (Figure 4).

Figure 4. Confocal inverted microscopy images of Primary smooth muscle from the myometrium of Ovis aries (PSMo24) cells. (a, b) Confirmation of the characteristic morphology of smooth muscle cells. (c) Nuclei stained with DAPI (blue). (d) Visualization of actin filaments (F-actin, green).

METHOD VI - EVALUATION OF THE ANTIOXIDANT CAPACITY OF BIOPRODUCTS IN PSMo24 CELLS USING DCFH-DA AND ABAP METHODS
1d 5h 25m
Culture PSMo24 cells as described in items Go to ,Go to and Go to . After reaching confluence, perform trypsinization as described in Go to .
Add 5.0 x 10x4 PSMo24 cells per well with Amount200 µL of culture medium Go to in black clear-bottom plates for fluorescence.
Incubate the plate at Temperature37 °C in a 5 % CO₂ incubator for Duration24:00:00 .

1d
Incubation
Overnight
After 24 hours, carefully invert the plate to remove the culture medium and wash with Amount100 µL of PBS to remove non-adherent or dead cells.
Apply the controls and treatments at the desired concentrations and incubate at Temperature37 °C in a CO₂ incubator with 5 % CO₂ for Duration04:00:00 or for the necessary time required for the applied treatment reaction.
4h
Incubation
Negative control: Untreated cells (DMSO, if used as a vehicle).
Positive control: Quercetin, solutions of 25, 50, 100, 175, and 250 µM Go to in serum-free medium (final solutions with <2 % DMSO). 

After the treatment exposure time, add DCFH-DA (60 µM Go to ) for Duration00:20:00 in an incubator at Temperature37 °C with 5 % CO₂.
20m
Wash the wells with PBS to retain only the antioxidants that entered the cells.
Add Amount100 µL of the ABAP solution (600 µM Go to ) and immediately place it in the microplate reader with a inverted fluorescence detector, programmed to take readings every Duration00:05:00 for Duration01:00:00 (a total of 13 readings). Measure fluorescence at 485 nm excitation and 538 nm emission.
1h 5m
Determine the antioxidant capacity based on the percentage decrease in fluorescence from the area under the curves generated by the 13 points provided by the reader, using the following formula:

Note
AAC (%) = 1 – AUC of the control / AUC of the sample* 100

Where:
AAC (%): percentage of antioxidant capacity (AAC) of the evaluated sample.
AUC of the control: Area under the curve of the control (without antioxidant).
AUC of the sample: Area under the curve of the sample (with antioxidant).


METHOD VII - MTT ASSAY (3-(4,5-DIMETHYLTIAZOL-2-YL)-2,5- DIPHENYLTETRAZOLIUM BROMIDE) TECHNIQUE FOR ASSESSING VIABILITY, CYTOTOXICITY, AND PROLIFERATION OF PSMo24 CELLS
1d 8h
Culture the PSMo24 cells as described inGo to and Go to . After confluence, perform trypsinization as described in Go to .
In a 96-well plate, resuspend the PSMo24 cells at a concentration of 5.0 × 10⁴ cells/well inAmount200 µL of culture medium Go to .
Incubate the plate at Temperature37 °C in a CO2 incubator for Duration24:00:00 ;

1d
Incubation
After 24 hours, observe the cell adhesion under the microscope.
Imaging
Remove the culture medium by carefully inverting the plate.
Apply Amount100 µL of the treatments of interest, diluted in serum-free culture medium Go to in quadruplicate in the 96-well plate.
Incubate the plate at Temperature37 °C in the CO2 incubator for Duration04:00:00 or for the necessary duration of the treatment reaction. After the incubation time, remove the treatments by carefully inverting the plate.
4h
Add Amount100 µL of the 0.3 mg/mL MTT solution per well and incubate the plate at Temperature37 °C in the CO2 incubator for Duration04:00:00 .
4h
After the incubation period, carefully invert the plate to remove the MTT reagent and addAmount100 µL of DMSO to each well.
Keep the plate at TemperatureRoom temperature , shielded from light, and take it for reading immediately.
Remove the plate lid and measure the absorbance of each well at 570 nm, including the blanks and negative controls.
Subtract the absorbance of each well by the average absorbance of the blank.
Calculate the cytotoxicity using the following equation:

Note
Cell Viability (%)= Abs. Sample - Abs. Control (-) / Abs. Control (+)-Abs. Control (-) x 100


Where:
Abs = Absorbance

METHOD VIII - RNA EXTRACTION PROTOCOL FROM PSMo24 CELLS
1h 28m
Add Amount1 mL of Trizol directly to the frozen cells and homogenize using a pipette (up and down motion) until the cells are completely dissolved in the Trizol. Incubate for Duration00:05:00 at TemperatureRoom temperature ;
5m
Pipetting
AddAmount0.3 mL of Chloroform and homogenize using a pipette. Incubate for Duration00:02:00 toDuration00:03:00 at TemperatureRoom temperature ;

3m
Pipetting
Centrifuge for Centrifigation12.000 x g, 4°C, 00:15:00 ;
15m
Centrifigation
After centrifugation, transfer the aqueous supernatant to a new 1.5 mL Eppendorf tube using a pipette, avoiding the interface. To make this easier, tilt the tube at a 90° angle;
Add Amount0.5 mL of Isopropanol to the aqueous phase and homogenize the tube;
Incubate TemperatureOn ice for Duration00:10:00 ;
10m
Centrifuge forCentrifigation12.000 x g, 4°C, 00:10:00 At this stage, observe the formation of the RNA pellet;
10m
Discard the supernatant from the pellet by inverting the Eppendorf tube;
Resuspend the pellet in Amount1 mL of 75 % Ethanol for the first wash;
Centrifuge for Centrifigation7.500 x g, 4°C, 00:05:00 and discard the supernatant using a pipette;
5m
Pipetting
Resuspend the pellet in Amount0.5 mL of 75 % Ethanol for the second wash and centrifuge (Centrifigation7.500 x g, 4°C, 00:05:00 );
5m
Dry the pellet for Duration00:05:00 to Duration00:10:00 , ensuring no alcohol droplets remain before proceeding to the next phase;
15m
Resuspend the pellet in RNase-free water (Amount20 µL -Amount50 µL , depending on the initial size of the pellet);
Use the Nanodrop spectrophotometer to quantify the RNA , with the 260/280 ratio > 1.80. This step allows precise quantification and purity assessment of the extracted RNA;

Analyze
Confirm the integrity of the RNA through electrophoresis and a concentrated agarose gel (Amount0.92 g of agarose diluted in Amount50 mL of buffer solution). The tank should be set to 110V and run for Duration00:20:00 . See Figure 5;

Figure 5: Agarose gel electrophoresis showing the quality and integrity of RNA extracted from Primary ovine smooth muscle from the myometrium of Ovis aries (PSMo24) cells. The molecular weight marker is indicated at the edges of the gel, and the samples represent different experimental conditions.

20m
Proceed to gene expression approach.

Note
The results obtained in these stages were sent for transcriptome analysis of both the in vivo explanted PSMo24 cells and the cryopreserved tissue. They will be analyzed and submitted for publication.

METHOD IX - PREPARATION OF MEDIA FOR IN VIVO AND CRYOPRESERVED MYOMETRIAL TISSUE EXPLANT
The preparation of the media used for tissue collection and explantation should be done the day before collection to ensure that all components are properly prepared and sterile. This step is crucial for the success of the collection and subsequent cell culture. To ensure an appropriate environment for cell growth and viability throughout the process, the following media should be prepared.
Sodium chloride (NaCl) 0.9 %

Composition: Sodium chloride (NaCl) 0.9 % - Sterile saline solution.

Preparation: Dissolve Amount9 g of NaCl in Amount1 L of sterile distilled water to obtain an isotonic saline solution 0.9 %. Filter the solution through a sterile filter and store it in the refrigerator (Temperature4 °C ).
Tissue washing medium - 0.9 % NaCl with 1 % Antibiotics

Composition: 0.9 % Sodium chloride (NaCl) and antibiotics (10,000 units penicillin and 10 mg streptomycin/mL – Pen-Strep).

Preparation: In a sterile bottle, add Amount99 mL of 0.9 % NaCl, then add Amount1 mL of antibiotics to achieve a final concentration of 1 %. Mix well and store in the refrigerator (Temperature4 °C ).
Transport Medium - 0.9 % NaCl with 1 % antifungal

Composition: 0.9 % Sodium chloride (NaCl) and antifungal (Amphotericin B).

Preparation: In a sterile container, add Amount99 mL of 0.9 % NaCl, then add Amount1.0 mL of antifungal to achieve a final concentration of 1 %. Mix well and store in the refrigerator (Temperature4 °C ).
Phosphate Buffered Saline (PBS) 1x (Ph07.4 ).

Composition: PBS (concentrated) powder for 1 liter of distilled or deionized water.

Preparation: Add the contents of the PBS envelope (9.07-10.0 g/L) to Amount1 L of distilled or deionized water. Mix well until the powder is completely dissolved. Filter the solution to ensure its purity. Store in the refrigerator (Temperature4 °C ).

Cell washing medium – PBS with 1 % Antibiotics

Composition: PBS 1x and Antibiotics (Pen-Strep);  

Preparation: In a sterile container, add Amount99 mL of 1x PBS, then add Amount1 mL of antibiotics to achieve a final concentration of 1 %. Mix well and store in the refrigerator (Temperature4 °C ).

Enzymatic digestion medium – Collagenase (Concentration1 mg/mL ):

Composition: Collagenase type I: Amount100 mg , PBS (1x)

Preparation: Dissolve Amount100 mg or Amount10 mg of type I collagenase in Amount100 mL or Amount10 mL of PBS, respectively, resulting in a Concentration1 mg/mL solution.
Culture Medium (DMEM + F-12 + FBS + Antibiotics):

Composition: DMEM (Dulbecco's Modified Eagle's Medium), F-12 (Ham's F-12 Nutrient Mix), FBS (Fetal Bovine Serum), Antibiotics (Pen-Strep).

Preparation: Mix 1:1 of DMEM and F-12 to obtain a balanced culture medium rich in essential nutrients for cell growth. Add 10 % FBS and 1 % Pen-Strep. Filter the medium through a sterile 0.22 µm filter and store at Temperature4 °C for no more than 24 hours.

Trypsinization Medium

Composition: Trypsin (0.5 % - 10x) and PBS.

Preparation: Mix 10 % trypsin with 90 % PBS. Prepare the medium on the day of use.
Examples of Preparation by Bottle Size:
25 cm² flask (5 mL):             0.5 mL of trypsin + 4.5 mL of PBS
75 cm² flask (10 mL):           1.0 mL of trypsin + 9.0 mL of PBS
182 cm² flask (25 mL):  2.5 mL of trypsin + 22.5 mL of PBS
Cryopreservation medium for PSMO24 cells

Composition: FBS, dimethyl sulfoxide (DMSO)

Preparation: In the FBS to be prepared, add 10 % DMSO, store in 15 mL Falcon tubes, and freeze atTemperature-20 °C until use.
Cryopreservation Medium for Myometrial Tissue (Vitrification)

Composition: Ethylene glycol (EG), sucrose (SAC), Fetal Bovine Serum (FBS), and DMEM.

Preparation: Prepare a solution containingConcentration6 Molarity (M) of ethylene glycol (EG), Concentration0.25 Molarity (M) of sucrose, and 10 % FBS, completing the final volume with DMEM.

Example:
Ethylene glycol (EG): 41.44 mL (6 M)
Sucrose: 8.56 g (0.25 M)
Fetal Bovine Serum (FBS): 10 mL (10 %)
DMEM: Complete to a final volume of 100 mL

Mix the solution well to ensure all components are fully dissolved and homogeneous. Filter the medium through a 0.22 µm sterile filter and store at Temperature4 °C for no more than 24 hours.
Thawing medium I

Composition: DMEM and sucrose 0.5 M – molecular weight: 342.3 g/mol/L

Preparation: In a sterile Falcon tube, dissolve Amount1.711 g of sucrose and complete to Amount10 mL with DMEM. Mix well to ensure homogeneity. Filter and store at Temperature4 °C .

Thawing medium II

Composition: DMEM and 0.25 M sucrose solution
Preparation: In a sterile bottle, dissolveAmount0.855 g of sucrose and complete up to Amount10 mL with DMEM. Mix well to ensure homogeneity. Filter and store at Temperature4 °C .

Formaldehyde 37 % without methanol

Composition: Formaldehyde 37 %, PBS (1x)
Preparation: Add Amount9.0 mL of 1X PBS. Pipette Amount1.0 mL of 37 % formaldehyde without methanol. Mix until homogeneous.

0.1 % Triton X-100 solution in 1 % BSA

Composition: Triton X-100, BSA, Milli-Q water.

Preparation: Weigh Amount0.1 g of BSA and dissolve in approximately Amount8 mL of Milli-Q water. Add Amount10 µL of Triton X-100 (0.1 % v/v). Complete the volume to Amount10 mL with Milli-Q water and mix well until the solution is homogeneous. Store in the refrigerator.

Solution of 0.3 % Triton X-100 in 1 % BSA

Composition: Triton X-100, BSA, Milli-Q water.

Preparação: WeighAmount0.1 g of BSA and dissolve it in approximately Amount8 mL of Milli-Q water. Add Amount30 µL of Triton X-100 (1 %). Complete the volume to Amount10 mL with Milli-Q water and mix well until the solution is homogeneous. Store in the refrigerator.

DAPI solution
Composition: 4′,6-diamidino-2-phenylindole (DAPI), 0.1 % Triton X-100-BSA

Preparation: Prepare the stock solution of DAPI according to the manufacturer's specifications. Dilute Amount1 µL of the DAPI stock solution in Amount10 mL of the 0.1 % Triton in BSA solution (1:10,000). Mix well and protect from light. Store in the freezer.
Phalloidin 488 Solution

Composition: Phalloidin 488, 0.1 % Triton X-100-BSA

Preparation: Prepare the stock solution of Phalloidin 488 according to the manufacturer's specifications. Add Amount25 µL of the Phalloidin 488 stock solution to Amount10 mL of the 0.1 % Triton X-100 in BSA solution (1:400). Mix well and protect from light. Store in the freezer.
Antifade solution

Composition: Glycerol (molecular biology), PBS (1x).

Preparation: Add Amount8 mL of glycerol, then add Amount2 mL of 1X PBS. Mix well until the solution is homogeneous. Store in the refrigerator.
Standard Quercetin Solution (1 mM)

Composition: Quercetin (3.022 mg for 10 mL of solution), solvent (DMSO or ethanol).

Preparation: Dissolve Amount3.022 mg of quercetin in DMSO or ethanol to make Amount10 mL of a 1 mM stock solution. Store in amber bottles in the refrigerator (Temperature4 °C ).
Quercetin Solution Dilutions (25, 50, 100, 175 e 250 µM)
Concentrations and volumes:
o 25 µM: 0.25 mL of stock solution + 9.75 mL of PBS.
o 50 µM: 0.5 mL of stock solution + 9.5 mL of PBS.
o 100 µM: 1 mL of stock solution + 9 mL of PBS.
o 175 µM: 1.75 of stock solution + 8.25 mL of PBS.
o 250 µM: 2.5 mL of stock solution + 7.5 mL of PBS.

Store the prepared solutions in the refrigerator (Temperature4 °C ), protected from light.
DCFH-DA Solution 60 µM

Composition: Stock solution of DCFH-DA 10 mM (dissolved in DMSO); PBS

Prepare the 10 mM DCFH-DA stock solution: DissolveAmount4.86 mg of DCFH-DA in Amount1 mL of DMSO. Mix until completely dissolved. Store in an amber tube or protected from light atTemperature-20 °C for up to 1 month. To prepare the working solution at 60 µM: Add Amount60 µL of the stock solution (10 mM) to Amount9.940 µL of PBS. Mix gently to homogenize.

Storage: The working solution should be used immediately or stored on ice for a maximum of 2 hours. Always protect the solution from light to avoid degradation.
ABAP 600 µM solution

Composition: ABAP (2,2′-azobis(2-methylpropionamidine) dihydrochloride), PBS.

Preparation: Weigh Amount1.44 mg of ABAP (corresponding mass for a 600 µM solution in 10 mL). Transfer the powder to a Falcon tube, prepared in a light-protected environment. Gradually add Amount10 mL of PBS (Ph07.4 ) and mix well until completely dissolved. Ensure that the solution is homogeneous.

Storage: The solution should be freshly prepared before use to ensure its stability. During use, keep the solution on ice and protected from light to prevent degradation.
RESULTS
It was possible to establish the protocol for the cultivation of PSMo24 cells, as well as the protocol for the cryopreservation of myometrial tissue for later cell explantation.
The data obtained demonstrated that cells derived from fresh myometrial tissue reached approximately 80 % confluence in the first passage, with an average of 16.7 ± 2.33 days for fresh tissue and 18 ± 0.86 days for cryopreserved tissue (P>0.05). For subsequent passages, from the second to the tenth, the average time to reach confluence was 4.10 ± 0.18 days and 3.43 ± 0.16 days for fresh and cryopreserved tissue, respectively (Figure 6). In the comparative analysis between the two types of explant and the different passages, a significant difference was observed at passage 3 (p < 0,05), However, based on the experience obtained in the laboratory, cultures can reach confluence within a range of 2 to 7 days, which is within the reference intervals observed (Figure 6).


Figure 6. Growth scheme of Primary smooth muscle from the myometrium of Ovis Aries (PSMo24) explanted from fresh (in natura) and frozen tissue until reaching 80% confluence.

The morphological analysis of PSMo24 cells was confirmed by confocal inverted microscopy, as shown in Figure 4 Go to . The use of phalloidin, a specific marker for actin fibers, highlighted the organization of the cytoskeleton, allowing the distribution and density of actin fibers within the cells to be observed. Additionally, DAPI staining, which specifically binds to DNA, was used to assess cellular organization and the structural integrity of the nuclei. Through this staining, it was possible to verify the preservation of nuclear morphology, indicating that the cells maintained their protection and structure after culture. These results suggest that PSMo24 cells exhibit a well-organized and intact cellular architecture, which is essential to ensure the quality of cultures and the reliability of experiments.
Figure 7 shows the quantification of cells per cm² during in vitro culture. In PSMo24 cells from fresh explants, an increase in cell number was observed during the first three passages, followed by stability in passages 3, 4, and 5. Starting from the sixth passage, there was a decrease in cell density, accompanied by a more random response in subsequent passages, with occasional increases in passages 8 and 10, interspersed by drops in cell numbers, as observed in passages 6 and 9.
Figure 7. Mean and standard error of the mean (SEM) of the total number of Primary smooth muscle from the myometrium of Ovis Aries (PSMo24) cell per cm² (left side of the graph). On the right, mean and SEM of the total number of PSMo24 cells in different flask sizes: small (25 cm²), medium (75cm²), and large (182 cm²), for cells from fresh tissue (blue) and cryopreserved tissue (red).

In PSMo24 cells derived from cryopreserved tissue explants, an increase in confluence was observed from the first to the second passage, showing a significant difference compared to the cells from fresh tissue explants (P < 0.05). However, in the subsequent passages, cell proliferation gradually decreased, with no significant differences compared to the cells from fresh tissue explants (P > 0.05).
Additionally, experiments conducted with the PSMo24 cells throughout the early passages showed satisfactory results, validating the developed experimental model (METHODS V to VIII).
The results shows that the proposed methodology for culturing primary smooth muscle cells and the protocol for cryopreserving myometrial tissue are viable and have potential for future studies involving reproductive biotechnology, pharmacology, and evaluation of bioproducts in animals and humans.
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Jimenez, C. R., Penitente-Filho, J. M., Torres, C. A. A., Medeiros, A. M., Silva, L. S. . Vitrification of bovine preantral follicles with dimethylsulfoxide and sucrose plus α-tocopherol
https://doi.org/10.1590/S0100-736X2016000300010
Segeritz, C. P., Vallier, L.. Cell culture: Growing cells as model systems in vitro
https://doi.org/10.1016/B978-0-12-803077-6.00009-6
Gadelha, C. A. G., Vargas, M. A., Alves, N. G.. Translational research and innovation systems in health: Implications on the biopharmaceutical segment
https://doi.org/10.1590/0103-11042019S210
Boerma M., Burton G. R., Wang J., Fink L. M., McGehee R. E. Jr, Hauer-Jensen M.. Comparative expression profiling in primary and immortalized endothelial cells: changes in gene expression in response to hydroxy methylglutaryl-coenzyme A reductase inhibition.
https://doi.org/doi: 10.1097/01.mbc.0000220237.99843.a1
Chalak M., Hesaraki M., Mirbahari S. N., Yeganeh M., Abdi S., Rajabi S., Hemmatzadeh F.. Cell Immortality: In Vitro Effective Techniques to Achieve and Investigate Its Applications and Challenges.
https://doi.org/10.3390/life14030417
Jaiswal A. N., Vagga A.. Cryopreservation: A Review Article
https://doi.org/10.7759/cureus.31564
Arora, M. . Cell Culture Media: A Review
//dx.doi.org/10.13070/mm.en.3.175
Volgin, D. V.. Animal Biotechnology: Tools and Techniques
https://doi.org/10.1016/B978-0-12-416002-6.00017-1
Barba-Ostria C., Carrera-Pacheco S. E., Gonzalez-Pastor R., Heredia-Moya J., Mayorga-Ramos A., Rodríguez-Pólit C., Zúñiga-Miranda J., Arias-Almeida B., Guamán L. P.. Evaluation of Biological Activity of Natural Compounds: Current Trends and Methods
https://doi.org/10.3390/molecules27144490
Nguyen, T. V., Trang, P. N., Kumar, A.. Understanding PFAS toxicity through cell culture metabolomics: Current applications and future perspectives
https://doi.org/10.1016/j.envint.2024.108620
Weiskirchen, S., Schröder, S. K., Buhl, E. M., Weiskirchen, R.. A Beginner's Guide to Cell Culture: Practical Advice for Preventing Needless Problems
https://doi.org/10.3390/cells12050682
Zhao, C. . Cell culture: in vitro model system and a promising path to in vivo applications
https://doi.org/10.1080/01478885.2023.2170772
Miller, M. A., Zachary, J. F.. Mechanisms and Morphology of Cellular Injury, Adaptation, and Death
https://doi.org/10.1016/B978-0-323-35775-3.00001-1
Acknowledgements
The authors are grateful toward financial support by Brazililian National Council for Scientific and Technological Development (CNPq) and by São Paulo Research Foundation (FAPESP).