This protocol describes a novel encapsulation method of ovarian follicles in fibrin alginate interpenetrating network for in vitro culture. Secondary follicle consists of a centrally located oocytes surrounded by several layers of somatic cells, which support oocyte development, follicle encapsulation and culture in a three-dimensional environment allows follicle expansion while supporting communication between multiple cellular compartments and maintaining this spherical structure of the follicle. In the first step of this protocol, isolate secondary follicles from 16 day old mouse ovaries for the drop method.
Rinse and transfer follicles to an encapsulation solution. Aspirate each follicle with fibrinogen alginate solution pipet into thrombin calcium solution, and allow beads to cross link for the paraform method. Transfer individual follicles into drops of fibrinogen alginate solution on a paraform coated glass slide and add thrombin solution to initiate F-A-I-P-N cross-linking.
Then transfer the encapsulated follicles to a growth media and image, the encapsulated follicle in the FA IPN. Since this system combines structural support with biological responsiveness to support the growth and development of immature follicles to produce eggs, it may be applied for culture cell aggregates to retain cell cell contacts without limiting expansion. Hello, my name is Lonnie Shea and through a collaboration with three wood drip, we've developed a culture system for the in vitro maturation of ovarian follicles.
And this culture study really reflects a true collaboration between bioengineering and reproductive biology. And today, the people who actually develop this culture system are gonna present the methods to you. First, Ariel Checkoff is a postdoctoral fellow in bioengineering who developed the biomaterial system.
Next is Minsu, who is a reproductive biologist in the Department of Obstetrics and Gynecology, who developed the handling procedures for the ovarian tissue. And together they will present to you the methods for actually encapsulating and culturing ovarian tissue. The main advantage of the described technique of existing methods, like the standard culture on the flat surface plastic is the, the fibrin alginate system supports three dimensional architectural of the follicle while presenting mechanically dynamic environment for the rapidly expanding follicle.
The dynamic mechanical microenvironment in the current man provide additional advantages when this technology is translated from mass model to the potential clinical application because in human follicle needs much more extensive growth in order to produce mature and fertilizer eggs For optimal results. Perform all dissections in L 15 based media for pH control on 37 degrees Celsius, heated stages for temperature control and on a clean bench to minimize bacterial contamination. Transfer freshly dissected ovaries into a new 35 millimeter Petri dish with one to two milliliters of maintenance media containing 0.1%collagenase and 0.1%DNAs and incubate for 15 minutes in a carbon dioxide incubator.
Wash samples in DM to remove collagenase and then transfer to a 35 millimeter Petri dish with fresh DM in order to isolate follicles from the ovary. Gently flick follicles away from the whole ovary using 2 28 5 8 gauge needles attached to insulin syringes. Remove as much stroma cells as possible without damaging the integrity of the follicle.
Dissect out 20 to 40 secondary follicles with a diameter of 130 to 150 microns per ovary. These follicles usually have two to three layers of granulosis cells. Now to an IVF dish, add one milliliter of mm in the central well and three milliliters of mm in the outer ring.
Transfer intact follicles with the pipette into the outer ring of the IVF dish. Keep this IVF dish inside the incubator at 37 degrees Celsius and 5%carbon dioxide. After all the follicles are collected.
Examine samples under a dissecting microscope with a magnification of five to eight times select follicles of 130 to 150 micron size that exhibit healthy morphology including two to three granulosis cell layers, no separation between oocyte and granulosis cells and intact and round oocytes. Transfer the healthy follicles into the center of another IVF dish for encapsulation. For the drop method of follicle encapsulation, add one milliliter of 50 international units per milliliter of thrombin solution into the middle well of an IVF dish thaw and keep fibrinogen stock solution on ice.
And bring the fibrinogen to room temperature right before encapsulation. Immediately before use. Prepare the fibrinogen alginate solution by mixing one times PBS 0.5%alginate and 50 milligrams per milliliter.
Fibrinogen at at a two to one to one ratio in a 1.7 milliliter sterile micro centrifuge tube. Gently vortex and centrifuge the slightly cloudy solution to decrease evaporation. Turn off the heating stage on the dissecting microscope in the corner of the outer ring of an IVF dish place.
90 microliters of fibrinogen alginate solution for encapsulation and a second drop of 10 microliters for a washing step. Transfer 10 to 15 follicles into the 10 microliter drop with a minimal amount of culture media, and quickly mix then with a minimal amount of solution, transfer all follicles into the 90 microliter drop and mix aspirate one follicle within five microliters of fibrinogen alginate solution using a 10 microliter tip and release the fibrinogen alginate solution together with the follicle into the thrombin solution in the middle of the IVF dish. Continue to repeat this step until all the follicles are encapsulated.
Let the beads stand for five to seven minutes to cross-link the gel in the thrombin calcium solution. The beads will become darker as fibrin is formed. Transfer the beads into the maintenance media starting with the darker beads.
First incubate for 15 to 30 minutes at 37 degrees Celsius and rinse off the remaining thrombin. Finally, transfer the beads into a culture plate and image. Immediately prepare the fibrinogen alginate solution by mixing 0.5%alginate and 50 milligrams per milliliter.
Fibrinogen at a one to one ratio in a 1.7 milliliter sterile micro centrifuge tube pipette 7.5 microliter drops of the fibrinogen alginate mixture onto the parfum coated glass. Slide with a three millimeter spacer and transfer one follicle into each drop. With a minimal amount of media.
Add 7.5 microliters of thrombin solution to each drop without touching or mixing the drop with the follicle because the gel forms almost instantaneously. Cover the gels with the second para film coated glass. Slide and place in a 100 millimeter dish upside down in an incubator for five minutes.
Transfer the sticky beads into maintenance media using forceps if necessary, and then to a culture well every two days. Image the cultured follicles using a light microscope and measure the follicle diameter with image J.Also replace half of the growth media with fresh Equilibrated growth media every two days After eight days of culture, the hydrogel should appear clear due to complete degradation of the fibrin components and the follicles can reach their final dimensions greater than 400 microns in diameter. Remove the growth media and add 100 microliters of 10 international units per milliliter.
Alginate lia in alpha mem to degrade the alginate. Place the plate in an incubator for 25 to 30 minutes. Then remove the follicles from the partially dissolved beads with a blunt end tip.
Transfer follicles to the outer ring of an IVF dish with dissection media to wash out the alginate liaise, and then transfer into the inner ring. Now transfer the antral follicles to the outer and then inner ring of the IVF dish with maturation media containing HCG and EGF incubate in a carbon dioxide incubator for 15 to 16 hours. Examine the cultures for cumulus cell expansion to remove the cumulus cell.
Add higher IDE solution to a final concentration of 0.1 milligram per milliliter and incubate for two to three minutes at 37 degrees Celsius with 5%carbon dioxide. Mouth pipette to free. Oocytes from the cumulus cells finally determine their maturation stage under the microscope.
Alginate is a natural biomaterial that is suitable for in vitro follicle culture due to its gentle ation and biochemical characteristics such as mesh size, controllable rigidity, and biological inertness. Small encapsulated follicles experience low compressive force in alginate in the beginning of the culture. However, as the follicle expands, the displaced volume in the bead is increasing, which results in greater compressive force in the opposite direction of follicle expansion.
The chains of individual polymers are completely entangled in fibrillin alginate solution prior to cross-linking. Both components of F-A-I-P-N start to cross-link immediately as they're exposed to the mixture of thrombin and a calcium follicles degrade the fibrin component of the F-A-I-P-N during the culture period, which is demonstrated by a clear circle around the follicle. The follicle 3D architecture is supported by the remaining alginate.
These images show secondary follicle growth in F-A-I-P-N over time from day 0 4 6, and eight to the resulting M two stage X.With polar body, a pronin is a protease inhibitor that can be added to the culture media to slow fibrin degradation. When 0.01 TIU per milliliter of aprotinin is added to the culture media in the first four days, follicles are not able to degrade the matrix. And after a protein in removal, the follicles resume growth and reach antral stage.
However, follicles cultured initially in a higher concentration of aprotinin, a growth inhibited. Today we have demonstrated a new technique for varying follicle encapsulation and in vitro culture in a three dimensional fibrin alginate network. The system not only maintains intact follicle structure through the culture, but also supports successful follicular genesis in vitro, which results in M two stage O site.
This technology is important to discover how the ovarian follicle works and has the potential to provide fertility options to young cancer patients More generally. This culture system can be used for the culture of cell clusters or the culture of cells as they organize into structures. These 3D culture systems will provide valuable tools to molecular dissect tissue growth.
