Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Summary

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Abstract

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine^1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules^4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold⁶. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

Protocol

1. Isolating Adipose-Derived Stem Cells (ASC)

Note: All procedures were performed at room temperature unless otherwise noted.

1. Isolate rat perirenal and epididymal adipose and wash with sterile Hank's buffered salt solution (HBSS) containing 1% fetal bovine serum (FBS) as previously described⁶.
2. Mince the tissue and transfer 1-2 g into 25 mL of HBSS containing 1% FBS into a 50 mL tube and centrifuge at 500 g for 8 min at room temperature.
3. Collect the free-floating adipose tissue layer and transfer to 125 mL erlenmeyer flask and treat with 25 mL of collagenase type II (200 U/mL) in HBSS for 45 min at 37 °C on an orbital shaker (125 rpm).
4. Carefully remove the liquid fraction (below oil and adipose layer) and filter sequentially through a 100-μm and 70-μm nylon mesh filter. Centrifuge the filtrate at 500 g for 10 min at room temperature, aspirate the supernatant and wash the pellet twice with 25 mL HBSS.
5. Resuspend the cell pellet in 50 mL of growth medium (MesenPRO RS Basal Medium) supplemented with MesenPRO RS Growth Supplement, antibiotic-antimycotic (100 U/mL of penicillin G, 100 μg/mL streptomycin sulfate, and 0.25 μg/mL Amphotericin B), and 2 mM L-glutamine and pipet cells into 2 T75 flasks (25 mL/flask).
6. Culture the ASC in a 5% CO₂ humidified incubator at 37 °C (Passage 2-4 ASC are used for all experiments).

2. Preparing Chitosan Microspheres (CSM)

Note: All procedures were performed at room temperature unless otherwise noted.

1. CSM are prepared by a water-in-oil emulsification process along with an ionic coacervation technique using our previous protocol⁶. Emulsify an aqueous solution of chitosan (6 mL of 3%w/v chitosan in 0.5 M acetic acid) in a 100 mL of an oil phase mixture consisting of soya oil, n-octanol (1:2 v/v) and 5% sorbitan-mono-oleate (span 80) emulsifier, using overhead (1700 rpm) and magnetic stirring (1000 rpm) simultaneously, in opposite directions. This dual method of mixing ensures that micelles formed early on before cross-linking occurs can remain in solution and not settle to the bottom. Furthermore, the magnetic stir bar aids in de-aggregating chitosan during micelle formation and rigidization.
2. The mixture is continuously stirred for approximately 1 hour until a stable water-in-oil emulsion is obtained. Ionic crosslinking is initiated with the addition of 1.5 mL of 1% w/v potassium hydroxide in n-octanol every 15 min for 4 h (24 mL total).
3. After completion of the crosslinking reaction, slowly decant the oil phase of the mixture containing CSM and immediately add the spheres to 100 mL of acetone. Wash the spheres with acetone until the oil phase is completely removed.
4. Dry the recovered spheres in a vacuum desiccator and analyze without further processing. The average CSM particle size, surface area per milligram and the unit cubic volume was determined using particle size analyzer.
5. For subsequent experiments, wash CSM three times with sterile water to remove residual salts and sterilize with absolute alcohol.

3. Determining the Number of Free Amino Groups in CSM

Note: All procedures were performed at room temperature unless otherwise noted.

1. Determine the number of free amino groups present in CSM after ionic crosslinking by using the trinitro benzenesulfonic (TNBS) acid assay of Bubins and Ofner⁷. Incubate 5 mg of microspheres with 1 mL of 0.5% TNBS solution in a 50 mL glass tube for 4 h at 40 °C and hydrolyze with the addition of 3 mL of 6N HCl at 60 °C for 2h.
2. Cool the samples to room temperature, and extract the free TNBS by adding 5 mL of deionized water and 10 mL of ethyl ether.
3. Warm a 5 mL aliquot of the aqueous phase to 40 °C in a water bath for 15 min to evaporate any residual ether, cool to room temperature, and dilute with 15 mL of water.
4. Measure the absorbance at 345 nm with a spectrophotometer using TNBS solution without chitosan as blank and the chitosan used for CSM preparation to determine the total number of amino groups. Estimate the number of free amino groups of the CSM relative to chitosan.

4. Loading ASC in CSM

Note: All procedures were performed at room temperature unless otherwise noted.

1. Equilibrate 5 mg of sterilized CSM from section 2.5 in sterile HBSS overnight and add to an 8-μm pore size membrane culture plate insert (24-well plate).
2. After the CSM have settled onto the membrane, carefully aspirate the HBSS and add 300 μL of growth medium to the inside of the insert and 700 μL of growth media to the outside of the insert.
3. Resuspend ASC at the appropriate concentration (1 x 10⁴ to 4 x 10⁴) in 200 μL of growth medium and seed over the CSM inside the culture plate insert. The final volume of medium within the culture insert, after seeding, is 500 μL.
4. Incubate the ASC seeded on CSM for 24 h in a 5% CO₂ humidified incubator at 37 °C.

5. Determining the Percentage of ASC Loading and Cell Viability in CSM

Note: All procedures were performed at room temperature unless otherwise noted.

1. After incubation, collect the ASC-loaded CSM in a sterile 1.5- ml microcentrifuge tube without disturbing the cells that have migrated into the insert membrane.
2. Remove the residual medium and add 250 μL of fresh growth medium to the tube.
3. To each tube add 25 μL MTT [3-(4,5-dimethylthiozole-2-yl)-2,5-diphenyltetrazolium bromide] solution (5 mg/mL) and incubate for 4 h in a 5% CO₂ humidified incubator at 37 °C.
4. After incubation, remove the medium, add 250 μL of dimethyl sulfoxide, and vortex the mixture for 2-5 min to solubilize the formazan complex.
5. Centrifuge the CSM at 2700 x g for 5 min, and determine the supernatant absorbance measured at 570 nm using 630 nm as reference.
6. Determine the cell number associated with the CSM relative to the absorbance value obtained from known numbers of viable ASC.

6. Characterizing ASC-CSM-Embedded Collagen Gel

Note: All procedures were performed at room temperature unless otherwise noted.

1. Mix ASC-loaded CSM (5 mg containing ≈ 2 x 10⁴ cells) with type 1 collagen (7.5 mg/mL) extracted from rat tail tendon according the method of Bornstein⁸ and fibrillate after adjusting the pH to 6.8 using 2 N NaOH.
2. Add the fibrillated collagen-ASC-CSM mixture to a 12-well plate and incubate for 30 min at in a 5% CO₂ humidified incubator at 37 °C.
3. After complete fibrillation, incubate the collagen-ASC-CSM gels for up to 14 days in a 5% CO₂ humidified incubator at 37 °C.
4. Release and migration of cells from CSM into the gel were observed using standard microscopy techniques.

7. Representative Results

In the present study, we have developed an in vitro strategy to deliver stem cells from chitosan microspheres (CSM) into a collagen gel scaffold. Porous CSM of uniform size (175-225 μm in diameter) and composition were prepared and used as cell carriers (Figure 2). Upon incubating ASC with the CSM, the cells attached at a concentration of 2 x 10⁴ cells/5mg of CSM. The cells were capable of spreading over the microsphere, while extending filopodia into the porous crevices of the microsphere (Figure 3). Once the cell-loaded CSM were mixed with the collagen gel, the cells immediately began migrating into the gels (Figure 4).

Table 1. Biological advantages for the use of chitosan and collagen in a stem cell delivery system.

Figure 1. Schematic depicting the overall strategy for the dual use of ASC-CSM loaded collagen scaffolds. Figures 2, 3, and 4 are annotated within the schematic to assist with interpretation of the images.

Figure 2. Schematic representation depicting the process of seeding stem cells onto chitosan microspheres. The process involves co-culturing ASC with CSM in an 8- μm pore size membrane culture plate insert. After 24 hours, the microspheres are removed from the insert and are ready for embedding into a biomaterial matrix.

Figure 3. Morphological characterization of CSM-loaded with ASC. Panel A depicts a light micrograph of ASC-loaded CSM, while panel B shows the same field of view superimposed with an image obtained by confocal fluorescence microscopy. ASC were preloaded with calcein AM (green). Panel C depicts an image of an SEM image of an unloaded microsphere, while panel D shows cells loaded onto the microsphere (asterisks). The TEM image in panel E shows a cross-section of an unloaded microsphere. A multitude of pores and crevices are located throughout the microsphere. Panel F shows a cross-sectioned microsphere with cells (arrows) attached and extending filopodia into the crevices. Original magnifications: A & B= 70X; C= 500x , D= 2,000x; E&F= 2,500x.

Figure 4. Migration of ASC from the CSM into the three-dimensional collagen scaffold. Panels A and B depict the CSM with cells migrating away from microsphere and into the collagen matrix on day 3 (A, arrows). Panel B shows a similar culture after 12 days. Transmission electron microscopy (TEM) images are depicted in C, D, and E. Asterisks in C and D show a microsphere that has been cross-sectioned with cells migrating away from the microsphere (arrows). Higher magnification of panel D is depicted in panel E, and shows cell filopodia attached to the collagen fibrils (inset). Original magnification: A&B = 100x; C&D = 6,000x; E = 20,000x, inset = 150,000x.

Figure 5. Schematic depicting the vast uses of CSM in regenerative medicine and drug delivery.

Discussion

A major hurdle in stem cell-based therapy is developing efficient methods for delivery of cells to the specified regions for repair. Due to patient to patient variability, the tissue type, injury size and depth; the methodology of delivering stem cells must be determined on a case-by-case basis. Although embedding stem cells within a matrix and delivering them to the wound site appears to be a next logical approach for tissue engineering, some technical hurdles remain. This includes the ability of the embedded cel...

Materials

Name	Company	Catalog Number	Comments
Name of the reagent/equipment	Company	Catalogue number	Comments
Hanks BalancedSalt Solution (HBSS)	Gibco	14175	Consumable
Fetal Bovine Serum	Hyclone	SH30071.03	Consumable
Collagenase Type II	Sigma-Aldrich	C6685	Consumable
70-μm nylon mesh filter	BD Biosciences	352350	Consumable
100-μm nylon mesh filter	BD Biosciences	352360	Consumable
MesenPRO Growth Medium System	Invitrogen	12746-012	Consumable
L-glutamine	Gibco	25030	Consumable
T75 Tissue Culture Flask	BD Biosciences	137787	Consumable
Chitosan	Sigma-Aldrich	448869	Consumable
Acetic Acid	Sigma-Aldrich	320099	Consumable
N-Octanol	Acros Organics	150630025	Consumable
Sorbitan-Mono-oleate	Sigma-Aldrich	S6760	Consumable
Potassium Hydroxide	Sigma-Aldrich	P1767	Consumable
Acetone	Fisher Scientific	L-4859	Consumable
Ethanol	Sigma-Aldrich	270741	Consumable
Trinitro Benzenesulfonic Acid	Sigma-Aldrich	P2297	Consumable
Hydrochloric Acid	Sigma-Aldrich	320331	Consumable
Ethyl Ether	Sigma-Aldrich	472-484	Consumable
8-μm Tissue Culture Plate Inserts	BD Biosciences	353097	Consumable
1.5-ml Microcentrifuge Tubes	Fisher	05-408-129	Consumable
MTT Reagent	Invitrogen	M6494	Consumable
Dimethyl Sulfoxide	Sigma-Aldrich	D8779	Consumable
Qtracker Cell Labeling Kit (Q tracker 655)	Molecular probes	Q2502PMP	Consumable
Type 1 Collagen	Travigen	3447-020-01	Consumable
Sodium Hydroxide	Sigma-Aldrich	S8045	Consumable
12-Well Tissue Culture Plates	BD Biosciences	353043	Consumable
Centrifuge	Eppendorf	5417R	Equipment
Orbital Shaker	New Brunswick Scienctific	C24	Equipment
Humidified Incubator with Air-5% CO2	Thermo Scientific	Model 370	Equipment
Overhead Stirrer	IKA	Visc6000	Equipment
Magnetic Stirrer	Corning	PC-210	Equipment
Vacuum Desiccator	-	-	Equipment
Particle Size Analyzer	Malvern	STP2000 Spraytec	Equipment
Water Bath	Fisher Scientific	Isotemp210	Equipment
Spectrophotometer	Beckman	Beckman Coulter DU800UV/Visible Spectrophotometer	Equipment
Vortex	Diagger	3030a	Equipment
Microplate Reader	Molecular Devices	SpectraMax M2	Equipment
Light/Fluorescence Microscope	Olympus	IX71	Equipment
Confocal Microscope	Olympus	FV-500 Laser Scanning Confocal Microscope	Equipment
Scanning Electron Microscope	Carl Zeiss MicroImaging	Leo 435 VP	Equipment
Transmission Electron Microscope	JEOL	JEOL 1230	Equipment