Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Summary

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.

Abstract

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.

Introduction

Increasing incidence of chronic diseases¹ has stimulated studies on transplantation of therapeutic cell populations². Syngenic or allogenic stem cells are the most commonly used cell types for these applications². However, these treatments do not allow control of differentiation and migration of stem cells after implantation and are not cost efficient. Transplantation of genetically modified cells with beneficial functions anticipates improving the treatment of many diseases. However, genetic cell modifications are recognized by the host’s immune system, therefore, these treatments require immunosuppression³. Encapsulation of cells producing insulin has been developed by Dr. Chang⁴. The technique is based on encapsulation of cells in alginate droplets that are immersed into a calcium chloride solution. Alginate molecules consist of mannuronic (M) and guluronic acid (G) and can be connected by Ca²⁺. After gelation, the beads are suspended a poly-L-lysine (PLL) solution. During this step, PLL binds to G and M in the alginate molecules which establishes the capsule’s membrane. The porosity of the capsule’s membrane can be modulated by varying the M and PLL concentrations, the incubation time, and temperature. The binding of PLL also depends on the type and concentration of alginate. Alginate matrices crosslinked with Ca²⁺ ions, are unstable in the physiological environment or in common buffer solutions with high concentration of phosphate and citrate ions. These buffers can extract Ca²⁺ from the alginate and liquefy the core. Liquefaction of the alginate core provides space inside the capsules for cellular movement and growth. Cells encapsulated in polyanionic alginate with polycationic poly-L-lysine (APL) are impermeable for immunoglobulins but have influx of nutrients and efflux of toxins. These APL's properties enable the long term survival of encapsulated cells after transplantation into genetically different hosts. Elliott et al. reported the survival of functioning encapsulated porcine pancreatic cells in a human patient nine years after implantation⁵.

Encapsulation techniques can be classified into microencapsulation (3-800 µm) and macroencapsulation (larger than 1,000 µm). Microcapsules are more durable than macrocapsules⁶. Since its discovery by Dr. Chang and colleagues in 1964, microencapsulation has been widely used for the encapsulation of anabolic cells producing insulin, other hormones, and bioactive molecules⁷. These treatments faced several challenges in the host tissue including fibrosis and immune response⁸. Initially, the side effects related to the quality of biopolymers have been resolved. However, transplantation of anabolic cells still initiates side effects, such as fibrosis, as a result of hormone overproduction outside of a specialized gland.

In recent decades, obesity and type 2 diabetes has reached epidemic proportions⁹. More than 30% of adult people worldwide are overweight and obese¹⁰. Increased intra-abdominal (iAb) fat formation increases incidence of chronic inflammation and promotes type 2 diabetes, cardiovascular disease, certain cancers, and other morbidities^11-13. Several lines of evidence suggested that pathogenesis associated with iAb fat can be averted by specific adipocytes. Recent studies have shown that transplantation of subcutaneous adipocytes into iAb region can improve metabolism and decrease obesity and insulin resistance in rodents in vivo¹⁴. Effective reduction of obesity and insulin resistance has been associated with thermogenic adipocytes capable of dissipating energy in form of heat^15,16. Thermogenic modification of adipocytes can be achieved by stable transfection of genes participating in the mitochondrial proton uncoupling, such as uncoupling protein 1 (Ucp1) or of genes regulating expression of Ucp1 and other thermogenic genes^15,16. Our recent studies showed that deficiency in aldehyde dehydrogenase 1 a1 (Aldh1a1) leads to the thermogenic remodeling of iAb fat that reduces obesity and insulin resistance in these mice^17,18. Notably, encapsulation of thermogenic Aldh1a1 deficient (Aldh1a1-/-) preadipocytes mediates same therapeutic effect in iAb fat in obese wild type mice, suggesting new therapeutic opportunities for treatment of iAb fat¹⁸. In experimental settings, encapsulated cells enable researchers to study effects of specific cell populations in a cost effective manner¹⁹. Here we discuss the method of encapsulation of a thermogenic catabolic cell line and its laboratory and therapeutic application in a mouse model of obesity. The protocol describes three phases for microcapsule production (Figure 1): the formation of the alginate microbeads (Figure 1A), the formation of the polycationic poly-L-lysine (PLL) membranes on the surface of microbeads (Figure 1B), and the removal of the alginate cores (Figure 1C).

Protocol

The study protocol was approved by The Ohio State University Ethics Committees. Animal experiments were approved by IACUC protocol. All procedures were performed under the level 2 biosafety cabinet with laminar flow. We followed all standard safety requirements and procedures. The microencapsulation technique for preparation of microcapsules has been performed as described^17,18.

1. Preparations of Materials

1. Prepare 10 ml of 2% sodium alginate solution in autoclaved physiological saline (0.9% NaCl in water). Prepare 0.05% PLL solution in physiological saline. Prepare these solutions the day before and stir overnight. Filter the solutions with 0.22 μm filter before use.
2. Prepare 50 mM sodium citrate and 100 mM CaCl₂ in 0.9% NaCl solutions and autoclave them.
3. Autoclave all solutions, needles, electrodes, and beakers. Thoroughly clean needles with wires to avoid clogging.
4. Determine the appropriate amount of cells to use for the capsules (102 microcapsules are needed for every cm² of well and there are approximately 500 cells per capsule¹⁸). Use 1 ml of sodium alginate per two million cells.
5. Prepare a ‘Fibroblast Growth Medium’ containing 10% calf serum, and 100 U/ml penicillin/streptomycin in a high glucose (4,500 mg/l glucose) Dulbecco's Modified Eagle's medium (DMEM).
   1. Prepare a ‘Differentiation Medium I’ containing 10% fetal bovine serum, 10 µg/ml insulin, 1 µM dexamethasone, 0.5 mM 3-isobutyl-1-methyl xanthine, and 100 U/ml penicillin/streptomycin in DMEM.
   2. Prepare a ‘Differentiation Medium II’ containing 10% fetal bovine serum, 10 µg/ml insulin, and 100 U/ml penicillin/streptomycin in DMEM.
6. Prepare lysis buffer containing one protease inhibitor tablet per 10 ml Radio-Immunoprecipitation Assay buffer (RIPA buffer).

2. Alginate Microbeads Preparation (Figure 1A)

1. Remove old medium from cell culture flask. Rinse cells with 10 ml of PBS. Bring cells in suspension with 0.25% trypsin-EDTA (2 ml per one confluent T175 flask).
2. Count cells in cell suspension using a hemocytometer. Use 10 ml aliquot from the cell suspension and count cells according to manufacturer’s instructions. Centrifuge the remaining cells in centrifugation medium at 480 x g at room temperature for 5 min.
3. Suspend the cell pellet in sodium alginate as described in step 1.4. Transfer the sodium alginate-cell solution to a 5 ml syringe.
4. Remove air bubbles in the solution, add a 23-gauge needle and invert the syringe to create a 1 ml pocket of air.
5. Place a small beaker (180 ml) containing 144 ml of 100 mM CaCl₂ solution under the needle spout of the encapsulator. Attach the electrode to the encapsulator with the tip approximately 2.5 cm above the surface of the 100 mM CaCl₂ solution.
6. Place tightly the syringe containing the sodium alginate-cell solution in the syringe pump. Attach the rubber tube to the opening of the syringe. Push the plunger until the sodium alginate-cell solution enters halfway through the tube. Adjust the voltage to 5.4 kV. Set the 12.06 mm diameter on the syringe pump. Adjust the speed to 3 ml/hr. Start the pump. Turn on the encapsulator and maintain the voltage at 5.4 kV.
7. Close the window of the hood and avoid any unnecessary vibration till the end of formation of the alginate microbeads. After all solution passes through the needle, solidify the alginate ball-shaped microbeads in the 100 mM CaCl₂ solution for additional 20 min before coating with PLL.

3. Coating Microbeads with PLL (Figure 1B)

1. Remove the beaker containing the sodium alginate-cell ball-shaped microbeads and transfer these microbeads into a 50 ml centrifugation tube. Remove CaCl₂ solution from the microbead pellet.
2. Wash the alginate-cell ball-shaped microbeads by adding 30 ml of 0.9% NaCl. Shake the tube by hand gently. Remove 0.9% NaCl with 25 ml pipette after the microbeads have precipitated by gravitation. Repeat two more times for a total of 3 washes.
3. Use 10 ml of 0.05% PLL solution for every 1 ml of sodium alginate solution. Add the 0.05% PLL and vortex at 1,000 rotations per min for 10 min.
   Note: Usually, 10 min is sufficient for PLL coating.
4. After PLL coat is formed, remove the PLL solution and wash the capsules 3 times as described in 3.2.

4. Removal of Alginate Core (Figure 1C)

1. Add 30 ml of 50 mM sodium citrate solution. Wait 5 min or until all the sodium alginate is dissolved. Wash capsules three times as described in step 3.1.1.
2. Remove the 0.9% NaCl, add 20 ml of culture medium to the 50 ml tube and transfer all capsules containing the cells to a cell culture flask. Handle encapsulated cells under standard cell culture conditions¹⁸.

5. In Vitro Applications to Study Xenograft and Host Cell Interactions or Kinetics of Metabolite Influx/Efflux between Cells (Figure 2)

1. Culture host cells on 24 well plate until confluent for co-cultures. Use the ‘Fibroblast Growth Medium’ for culturing preadipocytes.
2. Transfer microcapsules into 24 well plate containing confluent host cells. Add microcapsules to achieve a monolayer (102 microcapsules/cm² of well).
3. Induce pre-adipocyte differentiation with Differentiation Medium I. Every 48 hr, change media to Differentiation Medium II  for six days. Lyse cells in RIPA buffer. Use 50 µg protein per condition to analyze protein expression using Western blot.

6. In Vivo Application for Treatment of Obesity (Figure 3)

1. Mix 4% isoflurane with oxygen for induction of anesthesia and 2% isoflurane with oxygen for maintenance of anesthesia. Confirm adequate anesthetic depth by toe pinch.
   1. Apply anti-itch ointment on eyes to protect the corneas from drying out.
2. Use encapsulated cells that are permanently labeled with artificial fluorescence protein, such as green fluorescence protein (GFP).
3. Use a 3 ml syringe and a 20 gauge needle to inject encapsulated cells.
   1. Inject 0.5 x 10⁶ cells suspended in 0.2 ml of PBS into each iAb fat depot that is located in the intraperitoneal area between a gonad and kidney as shown in Figure 3. Use this volume of PBS and cell number for mice with an average weight of 40 g. Adjust cell number and volume in mice that have different weight or for determination of dose-dependent effects.

Results

Figure 1 shows that every step of microbeads production could be controlled under the microscope. Figure 2A shows how to co-culture adipocytes with a monolayer of encapsulated cells. Figure 2B is a representative example of a quantitative study using adipocyte/microcapsules co-cultures that were described in section 5. Lysates of adipocytes were analyzed using Western blot. Encapsulated cells were not analyzed in this experiment. Primary ATGL and β-actin antibodies were ...

Discussion

Various methods have been used to encapsulate cells, including drying, extrusion, and emulsion¹⁹. In this method, the alginate beads are extruded through a needle, then coated with PLL and the alginate core will be dissolved to complete the encapsulation. Although this method has been used for years, formation of the beads with the desired size and spherical shape is still challenging. The size of the capsules is highly dependent on the viscosity of sodium alginate solution, the extruder diameter and the dista...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Encapsulation device (VAR V1)	Nisco	LIN-0042	None
KD scientific syringe pump	KD scientific	780100Y	None
Olympus microscope	Olympus Optical	IX70-S8F2	None
Sodium alginate	Sigma	MKBP8122V	None
Poly-L-lysine hydrobromide (PLL)	Sigma	020M5006V	None
Calcium chloride	Sigma	SLBJ2662V	None
Sodium citrate tribasic dihydrate	Sigma	030M0200	None
Sodium chloride	Sigma	SLBD2595V	None
Mini-PROTEAN TGX Gels	Bio-Rad	456-1093	None
ATGL primary antibody (from rabbit)	Cell Signaling	2138S	None
Secondary anti body (anti rabbit)	LI-COR	926-68071	None
Radio-Immunoprecipitation Assay (RIPA) buffer	Boston BioProducts	D25Y6Z	None
Phosphate buffered saline (PBS)	Sigma	RNBD2893	None
Trypsin	Gibco	25200-056	None
Cortizone 10 anti-itch ointment	Cortizone 10	C4029138	None
Dulbecco's Modified Eagle Medium (DMEM)	Gibco	11965-092	None
Newborn calf serum (CS)	Sigma	N4762	None
Fetal bovine serum (FBS)	Sigma	F4135	None
3-Isobutyl-1-methylxanthine (IBMX)	Sigma	I0516	None
Dexamethasone	Sigma	D4902	None
Insulin (bovine)	Sigma	I5879	None
Protease inhibitor cocktail tablets	Roche	4693159001	None