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

Carolina Rodriguez Jimenez; Bruno Scatena Gatti; Livia Maria Presuto; Patricia Spoto Corrêa; Severino Matias de Alencar; Helder Louvandini

Apr 11, 2025

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

DOI

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

University of São Paulo

DOI: 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 25, 2025

Last Modified: April 11, 2025

Protocol Integer ID: 124907

Keywords: uterus, muscle, reproduction, sheep, culture

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. 

Attachments

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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
 

 

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 togo to step #9.2   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 togo to step #9.3   . 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 togo to step #9.5   ). Cut the tissue into smaller fragments of approximately Thikness0.5 cm ^2 , avoiding the collection of the caruncle area (Figure 1 Go togo to step #1.4   ).

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 togo to step #9.5   .     
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 togo to step #9.7   .    

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 togo to step #9.5   ). 
b. Medium replacement: Add Amount3-4 mL   of culture medium Go togo to step #9.7   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

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 PSMo₂₄CELLS-TRYPSINIZATION

1d 0h 14m

Once approximately 80 % confluence is reached, remove the culture medium and add Amount3 mL   of cell washing medium Go togo to step #9.5   .

Addition of Trypsin-EDTA: Add the trypsinization medium Go togo to step #9.8   : 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  .

Trypsin inactivation: Add Amount3 mL   of culture medium Go togo to step #9.7   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.

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  .

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

Part 1 (Criopreservation): Add Amount1 mL   of the cryopreservation solution Go togo to step #9.10    (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.

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.

METHOD III - SUBCULTURE AND THAWING OF PSMo₂₄ CELLS

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.

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 togo to step #9.7  .

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.

Add culture medium Go togo to step #9.7   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 PSMo₂₄CELLS

23m

Collection and Preparation of Myometrial Tissue, see item Go togo to step #1.1   to Go togo to step #1.8  .

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 togo to step #9.10  ) 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 togo to step #9.10    and immediately store in liquid nitrogen atTemperature-196 °C  .

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:

Wash I:
Place two tissue fragments in each well with Amount2 mL   of thawing solution I Go togo to step #9.11   and keep them for Duration00:05:00  . After this period, transfer the fragments to thawing solution II Go togo to step #9.12  .

Wash II:
Keep the fragments immersed in thawing solution IIGo togo to step #9.12   for Duration00:05:00  . Then, carefully transfer the fragments to thawing solution III (DMEM-Pure).

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 togo to step #9.5   and proceed with the enzymatic digestion, as mentioned in the item Go togo to step #1.9   . 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  .

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 togo to step #3  ,Go togo to step #3.2  and Go togo to step #4  . After confluence, perform trypsinization as described inGo togo to step #2  

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 togo to step #9.7  . Incubate the plate atTemperature37 °C  in a CO2 incubator for Duration24:00:00  .

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 togo to step #9.7  )  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.

Fix the sample in 3.7 % formaldehyde solution without methanol Go togo to step #9.13   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 togo to step #9.15   for Duration00:30:00  

30m

Incubate the cells in a dilution of: 1:10,000 of 4′,6-Diamidine-2´-phenylindole dihydrochloride (DAPI)  Go togo to step #9.16   and 1:400 of 1:400 of phalloidin 488  Go togo to step #9.17   , prepared in 1 % Triton X-100 in 1 % BSA  Go togo to step #9.14   . 

Add Amount100 µL   of a solution containing DAPI and Phalloidin 488 Go togo to step #9.17   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.

Wash once with PBS.

Add 1 drop per well of the antifade solution Go togo to step #9.18   and analyze within Duration48:00:00  

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

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 PSMo₂₄CELLS USING DCFH-DA AND ABAP METHODS

1d 5h 25m

Culture PSMo24 cells as described in items Go togo to step #3  ,Go togo to step #3.2   and Go togo to step #4  . After reaching confluence, perform trypsinization as described in Go togo to step #2  .

Add 5.0 x 10x4 PSMo24 cells per well with Amount200 µL   of culture medium Go togo to step #9.7   in black clear-bottom plates for fluorescence.

Incubate the plate at Temperature37 °C   in a  5 % CO₂ incubator for Duration24:00:00  .

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.

Negative control: Untreated cells (DMSO, if used as a vehicle).
Positive control: Quercetin, solutions of 25, 50, 100, 175, and 250 µM Go togo to step #9.19   in serum-free medium (final solutions with <2 % DMSO). 

After the treatment exposure time, add DCFH-DA (60 µM Go togo to step #9.20  ) 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 togo to step #9.21  ) 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 togo to step #3   and Go togo to step #4  . After confluence, perform trypsinization as described in Go togo to step #2  .

In a 96-well plate, resuspend the PSMo24 cells at a concentration of 5.0 × 10⁴ cells/well inAmount200 µL   of culture medium Go togo to step #9.7  .

Incubate the plate at Temperature37 °C   in a CO2 incubator for Duration24:00:00  ;

After 24 hours, observe the cell adhesion under the microscope.

Remove the culture medium by carefully inverting the plate.

Apply Amount100 µL   of the treatments of interest, diluted in serum-free culture medium Go togo to step #9.7    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.

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  .

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  ;

AddAmount0.3 mL  of Chloroform and homogenize using a pipette. Incubate for Duration00:02:00   toDuration00:03:00   at TemperatureRoom temperature  ; 

Centrifuge for Centrifigation12.000 x g, 4°C, 00:15:00 ;

15m

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;

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 );

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;

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 togo to step #5.12   . 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.

Protocol references

CITATION
Jimenez, C. R., Penitente-Filho, J. M., Torres, C. A. A., Medeiros, A. M., Silva, L. S.  (2016). Vitrification of bovine preantral follicles with dimethylsulfoxide and sucrose plus α-tocopherol. Pesquisa Veterinária Brasileira.LINK
https://doi.org/10.1590/S0100-736X2016000300010

CITATION
Segeritz, C. P., Vallier, L. (2017).  Cell culture: Growing cells as model systems in vitro. Basic Science Methods for Clinical Researchers .LINK
https://doi.org/10.1016/B978-0-12-803077-6.00009-6

CITATION
Zhao, C.  (2023). Cell culture: in vitro model system and a promising path to in vivo applications. Journal of Histotechnology.LINK
https://doi.org/10.1080/01478885.2023.2170772

CITATION
Boerma M., Burton G. R., Wang J., Fink L. M., McGehee R. E. Jr, Hauer-Jensen M. (2006). Comparative expression profiling in primary and immortalized endothelial cells: changes in gene expression in response to hydroxy methylglutaryl-coenzyme A reductase inhibition.. Blood Coagul Fibrinolysis.LINK
https://doi.org/doi: 10.1097/01.mbc.0000220237.99843.a1

CITATION
Chalak M., Hesaraki M., Mirbahari S. N., Yeganeh M., Abdi S., Rajabi S., Hemmatzadeh F. (2024). Cell Immortality: In Vitro Effective Techniques to Achieve and Investigate Its Applications and Challenges..LINK
https://doi.org/10.3390/life14030417

CITATION
Jaiswal A. N., Vagga A. (2022). Cryopreservation: A Review Article. Cureus.LINK
https://doi.org/10.7759/cureus.31564


CITATION
Arora, M.  (2023). Cell Culture Media: A Review. Methods Mater.LINK
//dx.doi.org/10.13070/mm.en.3.175

CITATION
Volgin, D. V. (2014). Animal Biotechnology: Tools and Techniques. Animal Biotechnology.LINK
https://doi.org/10.1016/B978-0-12-416002-6.00017-1

CITATION
Miller, M. A., Zachary, J. F. (2017). Mechanisms and Morphology of Cellular Injury, Adaptation, and Death. Pathologic Basis of Veterinary Disease.LINK
https://doi.org/10.1016/B978-0-323-35775-3.00001-1

CITATION
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. (2022). Evaluation of Biological Activity of Natural Compounds: Current Trends and Methods.  Molecules.LINK
https://doi.org/10.3390/molecules27144490

CITATION
Nguyen, T. V., Trang, P. N., Kumar, A. (2024). Understanding PFAS toxicity through cell culture metabolomics: Current applications and future perspectives. Enviromment International.LINK
https://doi.org/10.1016/j.envint.2024.108620

CITATION
Weiskirchen, S., Schröder, S. K., Buhl, E. M., Weiskirchen, R. (2023). A Beginner's Guide to Cell Culture: Practical Advice for Preventing Needless Problems. Cells.LINK
https://doi.org/10.3390/cells12050682

CITATION
Gadelha, C. A. G., Vargas, M. A., Alves, N. G. (2019). Translational research and innovation systems in health: Implications on the biopharmaceutical segment. Saúde em Debate.LINK
 https://doi.org/10.1590/0103-11042019S210

Citations

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).

Reagents	Company	Code
ABAP (Ácido 2,2'-azino-bis(3-etilbenzotiazolina-6-sulfônico), solução a 1 mM)	Sigma-Aldrich	440914
Amphotericin B Solution	Sigma-Aldrich	A2942
Antibiotic/Antimycotic	Sigma-Aldrich	A5955
Collagenase from Clostridium histolyticum	Sigma-Aldrich	C9891
DAPI - 4′,6-Diamidine-2′-phenylindole dihydrochloride	Sigma-Aldrich	D9542
DCFH-DA 2',7'- Dichlorofluorescin diacetate	Sigma-Aldrich	D6883
Dimethyl sulfoxide - DMSO	Sigma-Aldrich	D2650
Dulbecco’s Modified Eagle’s Medium/High Modified	Sigma-Aldrich	56436C
Ethylene glycol	Sigma-Aldrich	324558
Fetal Bovine Serum	Sigma-Aldrich	F7524
Formaldehyde solution	Sigma-Aldrich	818.708
Glycerol	Sigma-Aldrich	G7757
Ham' Nutrient Mixture F12	Sigma-Aldrich	51651C
Hanks’ Balanced Salt solution	Sigma-Aldrich	H6648
HEPES	Sigma-Aldrich	H6147
MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)	Sigma-Aldrich	CT01-5
penicilina/estreptomicina	Sigma-Aldrich	P4333
Phalloidin 488	Sigma-Aldrich	49409
Phosphate Buffered Saline	Sigma-Aldrich	806544
Quercetin Solution	Sigma-Aldrich	Q4951
Sacarose	Sigma-Aldrich	50389
Sodium Bicarbonate	Sigma-Aldrich	S5761
Sodium chloride	Sigma-Aldrich	S5886
Solução de azul de tripano a 0,4%.	Sigma-Aldrich	T6146
Triton X-100	Sigma-Aldrich	T8787
Trypsin-EDTA	Gibco	T2610
Absolute Ethanol	Êxodo Científica	AG07145RA
Isopropyl alcohol	Êxodo Científica	AI09965RA
Tri Reagent	Sigma-Aldrich	BCCB0163
Chloroform	Êxodo Científica	C09877RA

	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

	Equipment	Company
	Inverted Microscope	Bel photonics
	Heating Plate	Ted
	Flow 1 (Biological Safety Cabinet)	Pachane pa400eco
	Flow 2 (Biological Safety Cabinet)	Pachane pa70
	Refrigerator	Consul frostfree
	Freezer	Coldlab
	Incubator 1	Binder
	pH Meter 2 (Fixed)	Hanna edge pH
	Water Bath	Benchmark scientitic mybath 8l
	Balance	Bel engineering
	Magnetic Stirrer	Fisotom
	Ultrasonic Bath	Solidsteet
	Vortex Mixer	Nova

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

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