This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2013R1A1A2012159 and NRF-2013R1A1A2007561), Konkuk University, and the Department of Chemical Engineering and MCubed Program at the University of Michigan.

1. Whole-cell Biocatalysts Preparation &#160;
NOTE: The recombinant E. coli cells harboring pET28a-SpNox31 or pET28a-HjLAD28 are hereafter referred to as E. coliSpNox and E. coliHjLAD, respectively.
<ol>
	<li>Inoculate a single colony of E. coliHjLAD in 3 ml&#160;of Luria-Bertani (LB) medium supplemented with kanamycin (50 &#181;g/ml) and incubate in an incubator shaker O/N at 37 oC, 250 rpm.</li>
	<li>Dilute the culture by 1:100 in 200 ml&#160;of fresh LB containing 50 &#181;g/ml&#160;kanamycin and incubate at 37 oC, 250 rpm until the OD600 reaches ~0.6.</li>
	<li>Induce HjLAD protein expression by adding 0.1 mM isopropyl &#946;-D-thiogalactopyranoside (IPTG) to the culture medium and incubate at 16 oC, 180 rpm for 16 hr.
	<ol>
		<li>Alternatively, perform induction at 25 oC, 200 rpm for 6 hr&#160;if the L-xylulose biosynthesis (Step 2 below) is performed the same day.</li>
	</ol>
	</li>
	<li>Harvest the induced E. coliHjLAD cells by centrifugation at 3,200 x g for 20 min at 4 oC. Discard the supernatant and proceed to Step 2 to process the cell pellet.</li>
	<li>In parallel, perform steps 1.1 - 1.4 for E. coliSpNox.</li>
</ol>
2. Biosynthesis of L-xylulose by Coupling E. coliHjLAD and E. coliSpNox for Cofactor Regeneration
<ol>
	<li>Resuspend the cell pellets of E. coliHjLAD and E. coliSpNox separately in 50 mM Tris-HCl buffer (pH 8.0) at a cell density of 5.0 g dry cell weight (gDCW)/L. 
	Note: A correlation between gDCW and the optical density measured at 600 nm (OD600) can be established to facilitate the experiment. The formula used in this protocol is 1 gDCW/L=0.722*OD600 - 0.0965, which can vary among different spectrometers.</li>
	<li>Mix 600 &#181;l&#160;of 5.0 gDCW/L E. coliHjLAD, 600 &#181;l&#160;of 5.0 gDCW/L E. coliSpNox, 100 &#181;l&#160;of 20 mM NAD+, and 150 &#181;l&#160;of 2 M L-arabinitol in a 14&#160;ml&#160;round-bottom tube and bring the reaction volume to 2 ml&#160;by adding 550 &#181;l&#160;of 50 mM Tris-HCl (pH 8.0). 
	Note: The ratio of the two whole-cell biocatalysts amount can be optimized to improve the biosynthesis of the product. For the described system, a ratio of E. coliSpNox:E. coliHjLAD = 1:1 was found to be optimal for L-xylulose biosynthesis (Figure 1B).</li>
	<li>Incubate the reaction mixture at 30 oC, 200 rpm for 8 hr.</li>
	<li>Collect the supernatant after centrifugation at 4 oC, 3,200 x g for 10 min and proceed to quantify the L-xylulose production as described in Step 3 below.</li>
</ol>
3. Colorimetric Assay for L-xylulose Quantification
<ol>
	<li>Aspirate 100 &#181;l&#160;of the reaction supernatant collected from step 2.4 into a 1.5 ml&#160;tube.</li>
	<li>Add 50 &#181;l&#160;of 1.5% cysteine, 900 &#181;l&#160;of 70% sulfuric acid, and 50 &#181;l&#160;of 0.1 % carbazole dissolved in ethanol and mix gently by inverting the tube 3 times.</li>
	<li>Incubate the reaction mixture at 37 oC, 200 rpm for 20 min.</li>
	<li>Measure the optical absorbance of the reaction mixture at 560 nm (A560) using a spectrophotometer.
	<ol>
		<li>Dilute the reaction mixture if the A560 reading is above 1.</li>
	</ol>
	</li>
</ol>
4. Immobilization of Recombinant Whole-cell Catalysts in Calcium Alginate Beads
<ol>
	<li>Dissolve 4 g sodium alginate in 100 ml&#160;of distilled water. Prepare alginate solution by adding sodium alginate to water to avoid the formation of clumps. Heat the mixture if needed.</li>
	<li>Add 600 &#181;l&#160;of 5.0 gDCW/L E. coliHjLAD and 600 &#181;l&#160;of 5.0 gDCW/L E. coliSpNox in 1.2 ml&#160;4% alginate prepared in step 4.1 and mix the cells and alginate by gentle pipetting to avoid bubble formation.</li>
	<li>Aspirate the alginate/cell suspension into a syringe using a needle and add the mixture drop-wise into a 0.3 M calcium chloride (CaCl2) solution in a 100 ml&#160;beaker with continuous stirring. 
	Note: The volume of CaCl2 solution used for alginate bead formation should be enough for the alginate droplets to be completely submerged. Additionally, the distance between the syringe needle and the surface of the calcium chloride solution must be maintained within an optimal range to ensure formation of uniformly spherical beads. The optimum distance range can be determined experimentally and depends on the inner diameter of the needle. As an estimate, a distance of ~15 &#177; 5 cm was found to be optimal for a 0.6 cm (inner diameter) syringe needle.</li>
	<li>Leave the beads in the CaCl2 solution for 2 - 3 hr&#160;at RT without stirring to allow crosslinking and gel beads formation.</li>
	<li>Decant the CaCl2 solution without disturbing the beads by carefully pouring the CaCl2 solution into a 50 ml&#160;conical tube, and transfer the remaining beads in CaCl2 solution into another 50 ml&#160;conical tube.</li>
	<li>Wash the beads with 10 ml&#160;of 50 mM Tris-HCl (pH 8.0) buffer three times to remove excessive CaCl2 and un-encapsulated cells. 
	NOTE: At any given step, do not centrifuge the beads as doing so will rupture them. To separate the beads from solution, allow the suspension to stand undisturbed for 3 - 5 min. The beads will settle at the bottom and the wash buffer can be decanted into another container.
	<ol>
		<li>Do not discard the used wash buffer. Pool the used Tris-HCl wash buffer (30 ml) with the used CaCl2 solution collected from step 4.5.</li>
	</ol>
	</li>
	<li>Pellet the un-immobilized E. coli cells by centrifugation of the pooled CaCl2 and Tris-HCl solution collected from step 4.6 at 3,200 x g for 20 min. To determine the immobilization efficiency, calculate the density of the pelleted un-encapsulated cells in gDCW/L as described in step 2.1.</li>
	<li>Transfer the washed beads from step 4.6 into a tube. Follow steps 2.2 - 3.4 to evaluate the L-xylulose biosynthesis using all of the washed beads in place of cell pellets.</li>
</ol>
5. Stability Assay of Immobilized Biocatalysts for L-xylulose Production
<ol>
	<li>Collect the beads from step 4.8 and wash twice with 10 ml&#160;of 50 mM Tris-HCl (pH 8.0) buffer without centrifugation (as described in Step 4.6).</li>
	<li>Use all of the washed beads to perform the reaction as described in steps 2.2 - 3.4.</li>
	<li>Repeat steps 5.1 -&#160;5.2 for desired number of production cycles and measure the amount of L-xylulose produced in the reaction supernatant in each cycle.</li>
</ol>

The authors declare no competing financial interests. The paper aims at reporting detailed methodology to generate a coupled whole-cell biocatalytic system immobilized in alginate beads. Scientific novelties have been reported in a previous study16.

Recent technological advancements have enabled a surge in the commercialization of recombinant biotherapeutics, resulting in a gradual rise in their market value in the biotechnology industry. One such advancement is the advent of metabolic engineering in recombinant microorganisms, which has shown a great promise in establishing scalable industrial systems38. As with most processes, the successful commercialization of recombinant biomolecules produced by genetically engineered microbes is highly dependent on the production yield of the system39. This has led to the rapid development of &#34;brute force&#34; genetics39, where directed evolution of biocatalytic enzymes has been implemented in order to improve biocatalyst activity3. However, for certain metabolic pathways, such as redox pathways, mere biocatalyst improvement is not sufficient to influence economization of production owing to the significant effect of other essential reaction requirements, such as expensive cofactors. Scale-up of such a system, even with improved biocatalysts, will only serve to increase production costs. Thus, additional strategic methods are required for the commercialization of bioreactions involving stoichiometric ratios of cofactors. In addition, the recyclability of a biocatalyst is also necessary for improving the economic feasibility of a bioprocess. To overcome the limiting effect of rapid cofactor consumption and poor reusability of whole-cell biocatalysts, we have previously developed an immobilized, coupled whole-cell biocatalytic system for cofactor regeneration and stable re-use16. Described in the present study are the experimental procedures required to couple and co-immobilize two whole-cell biocatalysts for improved biotransformation yield and reusability, along with the representative results.
A critical factor to be considered for coupling the whole-cell biocatalysts is the maintenance of a precise control over the conditions established for protein induction and biocatalytic product formation to ensure reproducibility of the system. Variations in parameters like induction OD600, induction time, and IPTG concentration should be avoided to minimize batch-to-batch variation. Additionally, re-evaluation of the cell-to-cell ratio is required for different biotransformations. Note that the E. coliHjLAD:E. coliSpNox ratio of 1:1 reported in this study was determined by experimental optimization rather than the theoretical stoichiometric ratio. For immobilization of the recombinant whole-cell biocatalysts in calcium alginate beads, there are several critical steps to ensure uniformity with regard to the bead rigidity and distribution of cells. Firstly, the alginate solution must be prepared by slowly adding sodium alginate into water, and not vice versa to prevent clumping that could affect the local alginate concentration and thus the extent of cross-linking. Secondly, gentle pipetting should be employed when handling the cell-alginate suspension to avoid the formation of air bubbles, which inflict shear stress on the encapsulated cells, impede efficient mass transfer and can even cause the beads to float. In case air bubbles are introduced into the suspension, allow the cell-alginate suspension to stand undisturbed for 20 - 30 min&#160;to let the bubbles escape. Thirdly, when making the beads, the cell-alginate suspension must be added slowly and carefully in a drop-wise manner into a sufficient volume of calcium chloride solution for uniform size and morphology. Beads submerged incompletely in calcium chloride will have an irregular shape and poor rigidity that can contribute to cell leakage. Lastly, to avoid variability in the reaction volume from residual wash buffer, the beads in Step 4.8 can be lightly blotted with filter paper (do not air dry).
Common strategies for cofactor regeneration include co-culture of cells harboring functionally complementary enzymes and co-expression of these enzymes in the same cell. The described system employs a different strategy of coupling separate E. coliSpNox and E. coliHjLAD cell cultures, offering a better control in maintaining a desired ratio between the two enzymes. Additionally, cells expressing single proteins are subjected to less stress than those co-expressing multiple proteins, which could lead to reduced protein misfolding and increased protein expression40,41. Thus, direct whole-cell coupling presents the ability to mediate multi-enzyme catalytic processes without significantly compromising product yield. However, established strategies such as careful selection of bacterial promoters with varying strength42,43 and improvements in ribosome binding sites44 can be employed to mitigate the limited control over the ratio of target enzymes in co-culture or co-expression systems. Among the many immobilization techniques, encapsulation presents multiple advantages for immobilizing whole-cell biocatalysts over alternative methods such as cross-linking and adsorption, which are more suited for purified enzymes. Encapsulation of cells can also be performed in a variety of biomaterials like agar, chitosan, gums, carrageenan, gelatin and alginate45 for improving the reusability of the biocatalysts. However, calcium alginate encapsulation has been shown to be the most effective approach with respect to immobilization efficiency and biomolecule production46,47.
One major limitation of utilizing bacterial whole-cell biocatalysts is the minimal ability for post-translational modification of heterologous proteins in wild-type E. coli48. This restricts the successful use of eukaryotic biocatalysts within this system. An alternative to overcome this limitation could be the use of organisms such as yeast that are better equipped with eukaryotic post translational machinery. Another limitation of the described system is related to the use of immobilization for improved reusability. Immobilization is a common cost-effective strategy employed to counteract the poor stability of biocatalysts and improve their turnover rate by simplifying the bioprocess49-51. The main parameters that affect the catalytic performance of encapsulated whole-cell biocatalysts include sodium alginate concentration, CaCl2 concentration, cell loading and bead size. It is important to recognize that these parameters are not independent from each other. As such, a comprehensive analysis of all factors within a single study is very time-consuming, thus it is necessary to select the most crucial parameters for optimization. Evaluated and presented here are the effects of two parameters, sodium alginate and CaCl2 concentration. Although not discussed in detail here, we have previously performed the optimization of cell loading, comparing the dependence of yield on the weight of cells immobilized16. Reports in literature indicate that variation of bead size could also modulate the production yield by increasing enzyme encapsulation, improving activity via increased surface area of the immobilized system or by changing the diffusion of substrates and products due to altered transport properties. Depending on the biocatalytic reactions under consideration, selection of alternate critical factors may be required, which could lead to the generation of a different set of optimal conditions. A detailed study for establishment of optimal conditions for additional parameters provides scope for the further improvement in performance of the coupled whole-cell system.
In summary, we report the experimental implementation of a coupled whole-cell biocatalytic system immobilized in calcium alginate beads, illustrating the improved production of L-xylulose. By expressing different recombinant enzymes that facilitate other cofactor dependent biocatalytic pathways, high yields of many biologically relevant molecules can be obtained using the protocol described here. In addition, this system can be expanded to accommodate more than two recombinant biocatalysts for one-pot multi-enzyme biosynthesis of products52 and applications other than biocatalytic production, such as microbial biosensors for biochemical oxygen demand53. Taking advantage of the synergistic feature of this system, encapsulation of symbiotic microbial consortia can be used for a myriad of applications such as bioremediation54-56, whole-cell bioprocessing for biofuels57, plant growth promotion by soil bioaugmentation58 and biomining for metal recovery59. Taken together, the strategies of cofactor regeneration, multi-cell coupling and biocatalyst immobilization described herein can be used as a promising platform for improved production of important biomolecules pursued by others in the field.

Reductive whole-cell biotransformation using microorganisms has become a widespread method for the chemo-enzymatic synthesis of commercially and therapeutically important biomolecules1-3. It presents several advantages over the use of isolated enzymes, especially the elimination of cost-intensive downstream purification processes and the demonstration of an extended lifetime4-7. For biocatalytic pathways where cofactors are required for product formation, whole-cell systems have the potential to provide in situ cofactor regeneration via the addition of inexpensive electron-donating co-substrates5,8,9. However, this capacity is diminished for reactions that require a stoichiometric concentration of rare or expensive co-substrates10-13. Together with poor reusability of whole cells, this impedes the establishment of a scalable and continuous production system. Strategic modifications of whole-cell systems for these cofactor-dependent biotransformations are required to overcome the aforementioned limitations. Specifically, the combination of whole-cell biocatalysts that work cooperatively have been shown to significantly enhance the productivity and stability of the harbored enzymes14. These factors, which are often critical for enabling large-scale production of commercially viable products, can be optimized further by co-immobilizing biocatalytic microbes15. We have recently developed a simple and reusable whole-cell biocatalytic system that allows both cofactor regeneration and biocatalyst immobilization for the L-xylulose production16. In this study, this system was utilized as an example to illustrate the experimental procedures of applying these two strategies for improved biotransformation production yield and biocatalyst reusability.
L-xylulose belongs to a class of biologically useful molecules named rare sugars. Rare sugars are unique monosaccharides or sugar derivatives that occur very rarely in nature, but play crucial roles as recognition elements in bioactive molecules17,18. They have a variety of applications ranging from sweeteners, functional foods to potential therapeutics19. L-xylulose can be used as a potential inhibitor of multiple &#945;-glucosidases, and may also be used as an indicator of hepatitis or liver cirrhosis17,20. High efficiency conversion of xylitol to L-xylulose in whole-cell systems has been reported previously in Pantoea ananatis21,22, Alcaligenes sp. 701B23, Bacillus pallidus Y2524,25 and Escherichia coli26. In E. coli, however, it was achieved only using low (&#60;67 mM) xylitol concentrations26 due to potential inhibitory effects of an initial xylitol concentration higher than 100 mM on xylitol-4-dehydrogenase activity21,26. The thermodynamic equilibrium between xylulose and xylitol has been shown to strongly favor the formation of xylitol25,27. Additionally, xylulose yield is limited by the amount of expensive cofactors that have to be supplied in the absence of an in situ cofactor regeneration system. Together, these factors limit the potential for scaling into sustainable systems for L-xylulose biosynthesis.
To overcome these limitations and improve the L-xylulose biotransformation yield, the strategy of cofactor regeneration was employed first by establishing a coupled whole-cell biocatalytic system. Specifically, L-Arabinitol 4-dehydrogenase (EC 1.1.1.12) from Hypocrea jecorina (HjLAD), an enzyme in the L-arabinose catabolic pathway of fungi, was selected to catalyze the conversion of L-arabinitol into L-xylulose28,29. Like many biosynthetic enzymes, a major limitation of HjLAD is that it requires a stoichiometric amount of the expensive nicotinamide adenine dinucleotide cofactor (NAD+, the oxidized form of NADH) to carry out this conversion. NADH oxidase found in Streptococcus pyogenes (SpNox) has been shown to display high cofactor-regeneration activity30,31. Taking advantage of this attribute of SpNox, E. coli cells expressing HjLAD for the production of L-xylulose were coupled with E. coli cells expressing SpNox for the regeneration of NAD+ to boost the L-xylulose production depicted by the coupled reaction shown in Figure 1A. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, making this system much more efficient than those reported in literature.
The strategy of whole-cell immobilization was employed next to further enhance the reusability of the coupled biocatalytic system. Commonly used methods for whole-cell immobilization include adsorption/covalent linking to solid matrices, cross-linking/entrapment and encapsulation in polymeric networks32. Among these approaches, the most suitable method for cell immobilization is encapsulation in calcium alginate beads. Their mild gelation properties, inert aqueous matrix and high porosity help preserve the physiological properties and functionality of the encapsulated biologicals33. Therefore, the coupled biocatalyst system containing both E. coli cells harboring HjLAD or SpNox was immobilized in calcium alginate beads to enable multiple cycles of L-xylulose production (Figure 2).The immobilized biocatalyst system demonstrated good operational stability, maintaining 65% of the conversion yield of the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost its catalytic activity.

<table><tbody><tr><td>LB broth&amp;nbsp;</td><td>Sigma Aldrich</td><td>L3022-6X1KG</td><td></td></tr><tr><td>Kanamycin</td><td>Fisher</td><td>BP906-5</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-thiogalactopyranoside (IPTG)</td><td>Sigma Aldrich</td><td>I6758-10G</td><td></td></tr><tr><td>Tris base</td><td>Fisher</td><td>BP1521</td><td></td></tr><tr><td>B-Nicotinamide adenine dinucleotide hydrate</td><td>Sigma Aldrich</td><td>N7004-1G</td><td></td></tr><tr><td>L-Arabinitol</td><td>Sigma Aldrich</td><td>A3506-10G</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma Aldrich</td><td>168149</td><td></td></tr><tr><td>Sulfuric acid</td><td>Sigma Aldrich</td><td>320501-500ML</td><td></td></tr><tr><td>Carbazole</td><td>Sigma Aldrich</td><td>C5132</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Fisher</td><td>BP2818-4</td><td></td></tr><tr><td>Sodium alginate</td><td>Sigma Aldrich</td><td>W201502</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma Aldrich</td><td>223506-500G</td><td></td></tr><tr><td>Excella E24 shaker incubator</td><td>New Brunswick Scientific</td><td></td><td></td></tr><tr><td>Cary 60 UV-Vis Spectrophotometer</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>Centrifuge 5810R</td><td>Eppendrof</td><td></td><td></td></tr><tr><td>Beakers</td><td>Fisher</td><td></td><td></td></tr><tr><td>Syringe</td><td>Fisher</td><td></td><td></td></tr><tr><td>Needle</td><td>Fisher</td><td></td><td></td></tr><tr><td>Pioneer Analytical and Precision Weighing Balance</td><td>Ohaus</td><td></td><td></td></tr></tbody></table>

immobilization of multi-biocatalysts in alginate beads for cofactor regeneration and improved reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

To enable cofactor regeneration, L-xylulose synthesis was carried out in a coupled whole-cell biocatalytic system containing E. coliHjLAD and E. coliSpNox cells. Following the optimization of various parameters, the reusability of this system was improved by immobilizing it in calcium alginate beads (Figure 2).
L-xylulose Production with Cofactor Regeneration by Coupling E. coliHjLAD and E. coliSpNox cells. 
To enhance the bioconversion of L-arabinitol into L-xylulose, the E. coliHjLAD and E. coliSpNox cells were coupled to enable cofactor regeneration. To account for the differences in the protein expression and cofactor availability in E. coliSpNox and E. coliHjLAD cells, a comparison study of the L-xylulose yield at various E. coliSpNox-to-E. coliHjLAD ratios was performed. As shown in Figure 1B, the yield increased as the E. coliSpNox-to-E. coliHjLAD ratio increased from 0.13:1 to 1:1. The yields obtained for ratios 1:1 and 1.33:1 were equivalent and maximal for this coupled system. All further experiments were performed with the E. coliSpNox-to-E. coliHjLAD ratio of 1:1. Starting with an initial concentration of 150 mM L-arabinitol, the use of E. coliHjLAD biocatalyst alone produced 118 mM L-xylulose after 8 hr (saturation point for L-xylulose concentration), representing a yield of 79% (Figure 1C). In comparison, the use of the E. coliHjLAD and E. coliSpNox cells at a 1:1 ratio produced 144 mM L-xylulose, improving the yield to 96%.
Optimization of Whole-cell Biocatalyst Immobilization 
Immobilization of biocatalysts in calcium alginate beads offers many advantages, such as improved viability, as a result of the mild gel environment that protects the cells from the physical stresses in a bioreactor. The properties of these alginate beads is largely determined by formulation and processing parameters34. To optimize the production yield of L-xylulose by an immobilized form of the coupled whole-cell biocatalyst described above, two main parameters, namely sodium alginate concentration and CaCl2 concentration, were evaluated. These are the main parameters that determine the integrity and rigidity of the calcium alginate beads, which in turn affect the biocatalyst immobilization efficiency and molecular diffusion.
As shown in Figure 3A, the biocatalyst immobilization efficiency demonstrated an increasing trend with increasing sodium alginate concentration in the range of 1 - 3% (w/v). When the sodium alginate concentration used in the immobilization matrix was less than 1%, the resulting beads were fragile and easily broken due to low mechanical stability, releasing most of the cells into the surrounding CaCl2 solution (data not shown). Upon increasing the sodium alginate concentration to greater than 2%, the resulting beads hardened excessively leading to problems in molecular diffusion35 (Figure 4). Varying the CaCl2 concentration yielded a similar trend in the biocatalyst immobilization efficiency. For a fixed concentration of sodium alginate (2% w/v) and in the range of 0.1 - 0.4 M CaCl2, lower CaCl2 concentrations resulted in decreased rigidity of the beads and increased leakage of cells into the surrounding solution. This is due to the limited availability of Ca2+ ions for cross-linking the guluronate blocks on the alginate polymer chains36 (Figure 3B). However, increasing the CaCl2 concentration from 0.3 M to 0.4 M improved the biocatalyst immobilization efficiency only marginally. Due to the high immobilization efficiency (&gt;90%), CaCl2 concentrations beyond 0.4 M were not evaluated. When CaCl2 concentrations of less than 0.1 M were used, the sodium alginate droplet containing the biocatalyst fell apart in the CaCl2 solution and no spherical beads were formed (data not shown).
To maximize the biocatalyst immobilization efficiency without significantly slowing down the molecular diffusion in the calcium alginate beads, the sodium alginate and CaCl2 concentration were optimized next for improved L-xylulose production. Using the same concentration range as for immobilization efficiency (1 - 3% w/v sodium alginate and 0.1 - 0.4 M CaCl2), an optimal sodium alginate concentration of 2% was observed to achieve maximal yield of L-xylulose production (Figure 4A). Beyond this optimal concentration, the yield showed a decreasing trend. This was expected, owing to the issues in diffusion associated with the excessive rigidity of the calcium alginate beads. Similarly, the optimal CaCl2 concentration was found to be 0.3 M (Figure 4B). Combined with the data shown in Figure 3, the optimal sodium alginate concentration (2%) and CaCl2 concentration (0.3 M) that allowed the formation of beads with suitable rigidity and permeability was established, enabling minimal cell leakage and maximal L-xylulose production yield.
It should be noted that, another factor that affects the overall catalytic efficiency of the immobilized biocatalyst is the weight of the cell culture encapsulated. The catalytic efficiency increases with increasing cell inoculum up to an optimal inoculum weight, beyond which it shows a decreasing trend37. A possible explanation for this decrease is cellular overcrowding, which hinders molecular diffusion throughout the calcium alginate beads and decreases the catalytic efficiency of the system. Using the optimized immobilization condition above, the highest L-xylulose production was obtained with a cell loading of 3.75 (gDCW)/L16 (data not shown).
Reusability of the Immobilized and Free Whole-cell Biocatalysts for L-xylulose Production. 
Using the established optimized condition of 1:1 E. coliSpNox to E. coliHjLAD ratio, 2% (w/v) sodium alginate, and 0.3 M CaCl2, the immobilized coupled whole-cell biocatalyst output a maximal L-xylulose yield of 64%, compared to a much higher yield of 96% for the free coupled whole-cell system (cycle 1 of Figure 5). Note that the yield of immobilized E. coliHjLAD cell alone was only 32.5% (data not shown), lower than that of the immobilized coupled system as well as the free single biocatalyst system (79%, Figure 1C). This reduction was expected as immobilization is known to decrease the activity of biocatalysts, possibly due to substrate diffusion limitations, but was compensated by the advantage of improved reusability of a biocatalyst upon immobilization. This effect on the stability of the biocatalyst is demonstrated by the decreasing trend in L-xylulose yield observed on subjecting the free and immobilized systems repeatedly to a batch reaction process, shown in Figure 5. The yield for the free system showed a much steeper decline than that of the immobilized system, indicating a more rapid loss of enzyme activity in the free system. As a result, both systems had a comparable L-xylulose yield for production cycle 2. At the end of 7 cycles of successive re-use, the encapsulated biocatalysts maintained their stability and retained 65% of the original yield while the free whole-cell system retained less than 10% of its ability to produce L-xylulose. This demonstrates that the immobilized biocatalysts could be reused effectively for the production of L-xylulose and that the benefit of improved reusability and stability brought on by immobilization far outweighs the reduction in activity of the biocatalyst, conferring a significant advantage to the production process over a free whole-cell system.
<img alt="HJ-LAD/SpNox enzymatic process diagram; bar graphs of E. coli yield variations; L-xylulose yield." src="/files/ftp_upload/53944/53944fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Coupled Whole-cell System for the Biocatalytic Production of L-xylulose. (A) Schematic illustrating the cofactor regeneration reaction occurring in a coupled whole-cell system comprising E. coli cells harboring HjLAD or SpNox, respectively. (B) Variation in the yield of L-xylulose with different E. coliSpNox to E. coliHjLAD ratios. (C) L-xylulose yield from a whole-cell biocatalyst comprised of only E. coliHjLAD compared to that of a coupled whole-cell system comprised of E. coliHjLAD and E. coliSpNox cells capable of cofactor regeneration. The plots shown represent average and standard deviation of three independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53944/53944fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Sodium alginate immobilization process; syringe dispensing into CaCl2 solution; biocatalyst formation diagram." src="/files/ftp_upload/53944/53944fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Immobilization of the Whole-cell Biocatalysts via Encapsulation in Alginate Beads. Schematic representing the formation of uniformly sized Ca2+ alginate beads by dropwise addition of the sodium alginate-cell suspension to a solution of calcium chloride. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53944/53944fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Comparison of immobilization efficiency in bar charts; sodium alginate and CaCl2 concentration effects." src="/files/ftp_upload/53944/53944fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Parameters Influencing the Efficiency of Immobilization of the Whole-cell Biocatalyst in Alginate Beads. Immobilization efficiency as a function of (A) sodium alginate (%w/v) (B) CaCl2 (M) concentration. The plots shown represent the average and standard deviation of three independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53944/53944fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bar graph showing L-xylulose yield by sodium alginate concentration and CaCl2 molarity variations." src="/files/ftp_upload/53944/53944fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Evaluation of the Yield of L-xylulose from the Immobilized Coupled Whole-cell Biocatalyst as a function of (A) sodium alginate (% w/v) (B) CaCl2 (M) concentration. The plots shown represent the average and standard deviation of three independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53944/53944fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Chromatography result bar chart; relative yield vs. cycle number; experimental analysis." src="/files/ftp_upload/53944/53944fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5. Assessment of the Biocatalyst Reusability. The yield of L-xylulose production for 7 successive cycles of reuse of immobilized and free coupled whole-cell biocatalyst is shown in dark and light gray, respectively. The plot shown represents the average and standard deviation of three independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53944/53944fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability at JoVE.com

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

immobilization-multi-biocatalysts-alginate-beads-for-cofactor

Nanyang Technological University

Research

JoVE Journal

Chemistry

17.3K Views.  Konkuk University.  Weergegeven is het protocol voor co-immobiliserende hele cellen biokatalysatoren voor cofactorregeneratiesysteem en betere herbruikbaarheid, via de productie van L-xylulose als voorbeeld. De cofactor regeneratie wordt bereikt door het koppelen van twee Escherichia coli-stammen die functionele complementariteit enzymen; de hele cellen biokatalysator immobilisatie wordt bereikt door celinkapselende in calciumalginaat parels. 

Video: Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Developmental Biology

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

Bioengineering

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

Medicine

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Synthesis of Black Seed Oil-loaded-, Alginate-based, pH-sensitive Beads Using Electrospraying Technique

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in the context of colon cancer. Its active compound, thymoquinone, may help inhibit cancer cell growth and induce apoptosis in colon cancer cells. Additionally, black seed oil's anti-inflammatory and antioxidant effects could contribute to a healthier gut environment, potentially reducing cancer risk. Therefore, this study synthesized pH-sensitive alginate beads to deliver BSO into the colon in a controlled-release manner without releasing the drug at pH 1.2 (stomach), thus providing a well-defined release pattern at pH 6.8. The use of electrospray technology improves process performance by making it easier to formulate small, homogeneous beads with a higher rate of swelling and diffusion in the gastrointestinal medium. 
The formulated beads were characterized by an ex-vivo mucoadhesive strength test, bead size, sphericity factor (SF), encapsulation efficiency (EE), scanning electron microscope (SEM), in vitro swelling behavior (SB), and in vitro drug release in acidic and buffer media. All these manufactured beads demonstrated modest sizes of 0.58 &plusmn; 0.01 mm and spherical shape of 0.03 &plusmn; 0.00 mm in this testing. The formulation showed promising floating and releasing properties in vitro. With a very low cumulative percentage of beads, the oil EE of 90.13% &plusmn; 0.93% was high, and the release study demonstrated more than 90% in pH 6.8 with good floating nature in the stomach. Additionally, the beads were evenly spaced throughout the intestine. The electrospraying approach used in this protocol can be reproducible, yielding consistent outcomes. Therefore, this protocol can be used for large-scale production for commercialization purposes.

A technique employing high electric voltage and a targeted, active ingredient-loaded emulsion to fabricate pH-responsive, uniform microbeads is presented.

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in ...

Fabrication of Amyloid-&#946;-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic amyloid-&#946; (A&#946;) fragments, eventually leading to the classical features of the disease: amyloid plaques, neurofibrillary tangles and synaptic and neuronal loss. The lack of relevant non-transgenic preclinical models reflective of disease progression is one of the main factors hindering the discovery of effective drug treatments. To this end, we have developed a protocol for the fabrication of alginate microbeads containing amyloid-secreting cells useful for the study of the effects of chronic A&#946; production.
Chinese hamster ovary cells previously transfected with a human APP gene, secreting A&#946; (i.e., 7PA2 cells), were used in this study. A three-dimensional (3D) in vitro model for the sustained release of A&#946; was fabricated by encapsulation of 7PA2 cells in alginate. The process was optimized to target a bead diameter of 500-600 &#956;m for further in vivo studies. Optimization of 7PA2 cell encapsulation in alginate was performed altering fabrication parameters, e.g., alginate concentration, gel flow rate, electrostatic potential, head vibration frequency, gelling solution. Levels of secreted A&#946; were analyzed over time and compared between alginate beads and standard cell culture methods (up to 96 h).
A concentration of 1.5 x 106 7PA2 cells/mL and an alginate concentration of 2% (w/v) buffered with HEPES and subsequent gelation in 0.5 M calcium chloride for 5 min were found to fabricate the most stable microbeads. Fabricated microbeads were 1) of uniform size, 2) with an average diameter of 550 &#956;m, 3) containing about 100-150 cells per microbead and 4) able to secrete A&#946;.
In conclusion, our optimized method for the production of stable alginate microbeads containing amyloid-producing 7PA2 cells might enable the modeling of important aspects of AD both in vitro and in vivo.

This protocol illustrates a cell encapsulation method by rapid physical gelation of alginate to immobilize cells. Obtained microbeads allow controlled and sustained secretion of amyloid-&#946; over time and can be used to study the effects of secreted amyloid-&#946; in in vitro and in vivo models.

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic ...

Biology

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

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