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Encapsulation in fibrin-alginate hydrogel beads
<ol>
	<li>Prepare 1 mL of 50 IU/mL thrombin in Tris-buffered saline (TBS; pH 7.4) with 100 mM CaCl2&#160;in a 2.0 mL sterile microcentrifuge tube.</li>
	<li>Prepare fibrinogen stock solution (50 mg/mL in TBS) and keep it frozen. 
	NOTE: To avoid clumping when dissolving fibrinogen in TBS, heat TBS to 37 &#176;C.
	<ol>
		<li>On the day of the procedure, thaw it slowly on ice, and right before the use, bring to room temperature.</li>
	</ol>
	</li>
	<li>Just before use mix at a 1:1 ratio 0.5% alginate solution and 50 mg/mL fibrinogen solution to get finally 2 mL (FA). In a sterile microcentrifuge tube gently vortex the mixture. Avoid making bubbles.</li>
	<li>To prepare &#34;incubation chambers&#34;, apply thin strips of paraffin films to the glass microscope slides. 
	NOTE: Wipe the slides with 70 % EtOH before use. One incubation chamber is formed with two slides. To separate them, place 3 mm spacers on one of them.</li>
	<li>Pipette 7.5 &#956;L drops of the FA mixture onto this paraffin film-coated glass slide, which also has separating spacers. On one slide, place 8-10 drops, arranged in two rows.</li>
	<li>Transfer, using a micropipette, 3-5 cumulus oocyte complex (COCs) along with a minimum volume (up to 5 &#181;L) of maturation medium (MM), and place them precisely in the center of the FA drop. This procedure requires good manual skills and precision. &#160; 
	NOTE: The maturation medium (MM) consists of DMEM/F12, supplemented with 10 IU/mL PMSG, 10 IU/mL hCG, 10% FBS and 50% porcine follicular fluid (FF) collected in step 1.4.</li>
	<li>Add 7.5 &#956;L of thrombin/Ca2+&#160;solution on each FA drop, just to cover them. There is no need to mix them because the gel forms almost instantaneously.</li>
	<li>Cover the incubation chamber by applying the second, previously prepared glass slide to the FA capsules. With a very careful but firm movement, turn the chamber upside down and place it in a 100 mm Petri dish lined with moist filter paper to avoid drying.</li>
	<li>Then, transfer the Petri dish to the 5% CO2&#160;incubator (38 &#176;C) for about 5-7 min. After this, FA capsules will become cloudy because of simultaneous gelation of fibrin and alginate.</li>
	<li>Transfer FA capsules into 96 well plates (one capsule per well) containing 100 &#956;L MM per well. 
	NOTE: To not damage the formed FA capsules, perform this step carefully with the use of surgical forceps.</li>
	<li>Every 2 days replace half of the MM (50 &#956;L) with fresh, pre-equilibrated one. Image COCs using an inverted light microscope (at 10x magnification). 
	NOTE: Culture conditions for COCs FA culture: 38 &#176;C, under a 5% CO2&#160;atmosphere and relative humidity 95%. After 4 days of IVM, the hydrogels appear almost clear due to progressive degradation of the fibrin component. The remaining alginate should be degraded enzymatically - by alginate-lyase, a plant-derived enzyme that specifically degrades alginate and does not affect animal cells.
	<ol>
		<li>Remove MM from wells containing the capsules and add 100 &#956;L of 10 IU/mL alginate lyase in DMEM. Leave the culture plate in the incubator for 25-30 min.</li>
		<li>Remove COCs from the dissolved capsules. 
		NOTE: This can be done with a cut pipette tip.</li>
		<li>After several washes in a fresh DMEM, transfer COCs to the inner ring of an IVF dish (5-10 COCs per dish) containing PBS. Now, use them for further morphological or biochemical analysis.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>&lt;strong&gt;General&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Antibiotic Antimycotic (100x) 100ml</td><td>Thermo Fisher</td><td>15240062</td><td>2.5% final concentration for Handling Medium. 1% in PBS (step 1.2)</td></tr><tr><td>DMEM/F12 (500ml)</td><td>Sigma-Aldrich</td><td>D8062</td><td>Handling and Maturation Medium</td></tr><tr><td>DPBS (w/o Ca, Mg), 1x, 500ml</td><td>Thermo Fisher</td><td>14190144</td><td></td></tr><tr><td>FCS (100 ml)</td><td>Thermo Fisher</td><td>16140063</td><td>10% final concentration for both Handling Medium and Maturation Medium. (steps: 1.5. 2.6.)</td></tr><tr><td>PBS (1x, pH 7.4) 500ml</td><td>Thermo Fisher</td><td>10010023</td><td></td></tr><tr><td>TBS Stock Solution (10x, pH 7.4) 500 ml</td><td>Cayman Chemicals</td><td>600232</td><td>1x final concentration. Other brand can be use</td></tr><tr><td>&lt;strong&gt;Maturation Medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>hCG (1 VIAL of 10 000 U)</td><td>Sigma-Aldrich</td><td>CG10</td><td></td></tr><tr><td>PMSG</td><td>BioVendor</td><td>RP1782721000</td><td></td></tr><tr><td>Fibrin-alginate beads</td><td></td><td></td><td></td></tr><tr><td>Alginate Lyase</td><td>Sigma-Aldrich</td><td>A1603</td><td></td></tr><tr><td>Thrombin</td><td>Sigma-Aldrich</td><td>T9326-150UN</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C5670</td><td></td></tr><tr><td>Fibrinogen (250mg)</td><td>Sigma-Aldrich</td><td>F3879</td><td></td></tr><tr><td>Sodium Alginate</td><td>Sigma-Aldrich</td><td>W201502</td><td>&amp;nbsp;Use for alginate solution</td></tr><tr><td>&lt;strong&gt;Specific instruments, tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>30 mm Pteri dish</td><td>TPP</td><td>93040</td><td></td></tr><tr><td>60 mm IVF Petri dish</td><td>Falcon</td><td>353653</td><td></td></tr><tr><td>Ez-Grid Premium Cell Handling Pipettor</td><td>RI Life Sciences</td><td>8-72-288</td><td></td></tr><tr><td>Ez-Tip</td><td>RI Life Sciences</td><td>8-72-4155/20</td><td></td></tr><tr><td>Heating Table</td><td>SEMIC</td><td></td><td>Other brands can be used</td></tr><tr><td>Incubator</td><td>Panasonic</td><td>MCO-170AIC-PE</td><td>Other brands can be used</td></tr><tr><td>Sterile petri dish (10 cm)</td><td>NEST Biotechnology</td><td>704002</td><td></td></tr><tr><td>Sterile syringe filters with 0.2 &amp;micro;m</td><td>GOOGLAB SCIENTIFIC</td><td>GB-30-022PES</td><td></td></tr><tr><td>Thermos</td><td>Quechua</td><td>5602589</td><td>Other brands can be used</td></tr></tbody></table>

3d culture of porcine cocs: a procedure to encapsulate cumulus oocyte complexes in fibrin-alginate polymer hydrogel beads

Source: Gorczyca, G., et al. <a href="https://www.jove.com/v/61325">Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation.</a> J. Vis. Exp (2020).
This video describes the protocols for encapsulating porcine cumulus-oocyte complexes (COCs) in fibrin-alginate beads to establish a 3D culture.&#160; This in-vitro maturation system maintains normal cell-to-cell communication, ensuring oocyte maturation and retaining viability.

3D Culture of Porcine COCs: A Procedure to Encapsulate Cumulus Oocyte Complexes in Fibrin-alginate Polymer Hydrogel Beads

Source: Gorczyca, G., et al. Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation. J. Vis. Exp ...

The cumulus-oocyte complex, or COC, consists of a centrally located oocyte surrounded by closely arranged clusters of cumulus cells. To establish a 3D culture of porcine COCs, begin with a mix of alginate and fibrinogen biopolymers in the desired ratio.&#160;
Pipette drops of this mix onto a wax-coated glass slide. Seed this drop with pre-prepared COCs present in a suitable maturation media. This step ensures an adequate supply of nutrients from the media to the COCs.&#160;
Cover the polymer drop with the crosslinking solution containing thrombin and calcium ions. Place the spacers in the corners of the COC-containing slide and cover it with a fresh wax-coated slide to avoid shearing the polymer beads. Invert this slide assembly onto a petri dish and incubate.&#160;
During incubation, thrombin cleaves the fibrinogen into shorter fibrin fragments, which intercalate between the alginate polymer strands with the help of calcium ions. This results in the formation of a three-dimensional, interpenetrating polymer network, or IPN, encapsulating the COCs. Incubate the COC-polymer beads in the maturation media for a prolonged duration.&#160;
The IPN maintains oocyte-to-cumulus cells communication. This facilitates cytoplasmic differentiation involving organelle redistribution in the oocyte, which generates mature oocyte within the COC. Treat COC-polymer beads with the alginate-lyase enzyme to dissolve the IPN capsule, which then releases mature COCs. Transfer mature COCs to a fresh dish for further analysis.

3d-culture-porcine-cocs-procedure-to-encapsulate-cumulus-oocyte

Research

JoVE Encyclopedia of Experiments

Biology

Large Animal Models

Porcine

1.0K 次观看. 来源:Gorczyca， G. 等人 用于猪卵母细胞封装的蛋白水解降解海藻酸盐水凝胶和疏水微生物反应器。J. Vis. Exp （2020 年）。 本视频介绍了将猪卵丘-卵母细胞复合物 （COC） 封装在纤维蛋白-藻酸盐微珠中以建立 3D 培养的方案。这种体外成熟系统可维持正常的细胞间通讯，确保卵母细胞成熟并保持活力。 

视频: 猪 COCs 的 3D 培养:将积云卵母细胞复合物封装在纤维蛋白-海藻酸盐聚合物水凝胶珠中的程序 - 概念

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

三维脊髓前庭和听觉感官器官的培养

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

科研

JoVE Journal

发育生物学

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

医学

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

牛卵巢皮质组织培养

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

3D Culture of Porcine COCs: A Procedure to Encapsulate Cumulus Oocyte Complexes in Fibrin-alginate Polymer Hydrogel Beads

成績單

3D Culture of Porcine COCs: A Procedure to Encapsulate Cumulus Oocyte Complexes in Fibrin-alginate Polymer Hydrogel Beads