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Endocrinology, doi:10.1210/en.2003-0411
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Endocrinology Vol. 144, No. 11 4975-4979
Copyright © 2003 by The Endocrine Society

Microencapsulation of Leydig Cells: A System for Testosterone Supplementation

Marcelle Machluf, Anna Orsola, Stephen Boorjian, Richard Kershen and Anthony Atala

Laboratory for Cellular Therapeutics and Tissue Engineering, Children’s Hospital and Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Anthony Atala, M.D, 300 Longwood Avenue, Boston, Massachusetts 02115. E-mail: a.atala{at}tch.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The use of testosterone supplementation for elderly men has increased markedly over the last decade due to a recognized gradual decline in serum testosterone, which may lead to decreased bone mass, muscle strength, and libido. Testosterone supplementation is also used widely to treat some forms of erectile dysfunction, androgen deficiency, and infertility. However, long-term exogenous testosterone therapy has been associated with several complications, such as fluid retention, nitrogen retention, and hypertension. Due to these problems, alternate treatment modalities, involving more physiological and longer-acting systems for androgen delivery, have been pursued. Alginate-poly-L-lysine-encapsulated Leydig cell microspheres were used as a novel method for the delivery of testosterone in vivo. Encapsulated Leydig cells, which were stimulated with human chorionic gonadotropin, secreted high levels of testosterone in culture. Unencapsulated cells injected ip or sc failed to produce any testosterone levels, even with human chorionic gonadotropin stimulation. Castrated rats that were administered encapsulated Leydig cells ip or sc maintained a serum testosterone level between 0.23 and 0.51 ng/ml. Similar levels of testosterone were obtained for 43 d when the encapsulated Leydig cells were injected sc (0.28–0.48 ng/ml). Approximately 10% of a normal adult rat Leydig cell population was injected into each castrated animal; however, this resulted in serum testosterone levels of up to 40% of normal. Clinically, testosterone is usually delivered for supplementation and not for full replacement therapy. Therefore, the findings of this study suggest that microencapsulated Leydig cells may be a viable option as a therapeutic modality involving testosterone supplementation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGEN REPLACEMENT THERAPY is given to patients with testicular dysfunction and hypogonadal disorders to restore and maintain physiological levels of serum testosterone and its metabolites, dihydrotestosterone and estradiol. Androgen therapy may increase muscle strength, stabilize bone density, improve osteoporosis, and restore secondary sexual characteristics, including libido and erectile function (1). Currently available androgen replacement modalities include the oral administration of testosterone tablets or capsules, depot injections, and skin patches (2, 3, 4).

Long-term exogenous testosterone therapy has been associated with several complications, such as fluid and nitrogen retention, erythropoiesis, hypertension, and bone- density changes (5). In addition, fluctuating serum testosterone levels may occur, and frequent treatments may be required. Due to these problems, alternate treatment modalities, involving more physiological and longer-acting systems for androgen delivery, have been pursued.

The Leydig cells of the testes are the major source of testosterone in men. Implantation of heterologous Leydig cells or gonadal tissue fragments has been proposed as a method for chronic testosterone replacement (6, 7). However, these approaches were limited by the failure of the tissues and cells to produce testosterone.

Cell encapsulation in biocompatible and semipermeable polymeric membranes has been an effective method for immunoprotection, regardless of the type of recipient (allograft, xenograft, etc.) (8, 9). Encapsulated cells can maintain their viability while allowing for the secretion of desired therapeutic agents, either continuously or in response to specific physiologic stimulations. The majority of the implantation work using microencapsulated cells as delivery vehicles has employed two polymeric materials, sodium alginate and poly-L-lysine (PLL) (10, 11). Alginate microcapsules have been used for several applications, including pancreatic islet cells for insulin delivery and recombinant cells for the delivery of therapeutic gene products (10, 12, 13).

In the present study, we report the use of alginate-PLL-encapsulated Leydig cell microspheres as a novel method for the delivery of testosterone in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Leydig cell isolation
National Institutes of Health guidelines and institutional approval for the care and use of laboratory animals were observed. Male Sprague Dawley rats, 56–70 d old, were anesthetized using a mixture of 1:1 ketamine (45 mg/kg)/xylazine (10 mg/kg). Testes were immediately removed and placed on ice in M199 medium-buffered sodium bicarbonate and BSA. Leydig cells were isolated from 20 testes for each experiment. Each testis was washed three times with cold M199-BSA, decapsulated, and suspended in 8 ml of M199-BSA containing 0.25 g/ml collagenase (type IV; Sigma Chemical Co., St. Louis, MO) and soybean trypsin inhibitor (0.5 µg/ml; Life Technologies, Inc., Gaithersburg, MD). The tubes with the testes were shaken for 20–25 min in a 37 C water bath. The digested testes were diluted to 50 ml with cold M199-BSA and allowed to settle for 10 min at room temperature. The supernatants were filtered sequentially through sterile 140-µm and 73-µm steel meshes (Sigma). Cell pellets, formed after 5 min of centrifugation (120 x g), were washed twice with M199-BSA and layered at a concentration of 10 x 105 cells over each 35-ml Percoll gradient (1.077 g/ml; Amersham Biosciences, Uppsala, Sweden). Cells containing Percoll were centrifuged for 20 min (800 x g) at 4 C. For each Percoll run, an additional tube containing only marker beads with a known density (Pharmacia Biotech, Piscataway, NJ) was centrifuged. Cells from band III (a total of four bands) were collected from each tube, diluted with M199-BSA, and centrifuged at 150 x g for 15 min. The pellets were washed twice with M199-BSA and centrifuged for 10 min in 80 x g.

Leydig cell culture
Leydig cells were plated at a density of 3 x 106 cells/well in M199 buffered with 2.2 g/liter sodium bicarbonate and 1% fetal bovine serum. Cells were incubated at either 32 C or 37 C with 5% CO2. Cells were supplemented with endothelial growth factor (2 ng/ml), insulin, and a basal level of human chorionic gonadotropin (hCG; 2.5 x 10-4 IU). In all experiments, cell viability was measured using trypan blue and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT).

Leydig cell characterization
The Leydig cells were characterized by the following two methods: by using a 3ß-hydroxysteroid dehydrogenase (3ß-HSD) reaction assay (14) and by immunostaining with 3ß-HSD antiantibodies. Immunohistochemistry was performed using the Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA). Cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min. Cells were incubated with a rabbit polyclonal antibody against 3ß-HSD (a gift from Dr. Van Luu-The, University of Laval, Quebec, Canada) in a dilution of 1:500. The cells were incubated with a biotin-labeled secondary antibody and an avidin-biotinylated horseradish peroxidase complex (Vector). Positive staining was detected using a 3,3' diaminobenzidine tetrahydrochloride chromogen.

Testosterone production
Cells were stimulated with hCG (0.04 IU) every 24 h. The cell medium was sampled at 4, 8, 12, and every 24 h. In a second set of experiments, the cell medium was changed every 24 h before hCG stimulation. Two sets of control experiments were performed. As a first control, cells were not cultured with a basal level of hCG or stimulated with hCG. As a second control, the cells were maintained on a basal level of hCG but were not stimulated with hCG. The sampled media were stored immediately at -20 C until testosterone analyses. Testosterone levels were measured using a solid-phase RIA (Diagnostic Products Corp., Los Angeles, CA).

Leydig cell microencapsulation
Isolated Leydig cells were encapsulated within microspheres composed of Ca-alginate, were coated with the positively charged polyelectrolyte PLL, and were recoated with alginate (15). Briefly, pellets of Leydig cells were resuspended in sodium alginate-in-saline (1.2% wt/vol, Pronova UP MVG; Pronova Biomedical, Oslo, Norway) to a final ratio of 3 x 106 cells/1 ml of alginate. The suspension was sprayed through a 22-gauge needle located inside an air jet-head droplet-forming apparatus into a solution of HEPES-buffered calcium chloride [13 mM, 1.5% (wt/vol) CaCl2, pH 7.4; Sigma], where it gelled for 20 min (10, 11). The cell-Ca-alginate beads were washed three times with HEPES to remove free calcium ions. The alginate microspheres were coated with 0.1% (wt/vol) PLL of 21-kDa molecular mass (MM; Sigma) in saline for 12 min with gentle agitation. Unreacted PLL was removed by washing the microspheres three times with 13 mM HEPES-buffered saline, and the microcapsules were additionally coated with 0.125% sodium alginate for 10 min.

Microencapsulated Leydig cell testosterone production in vitro
The microencapsulated Leydig cells were suspended in M199-fetal bovine serum medium at an approximate density of 3 x 106 cells/well. Microcapsules were incubated at 32 C or 37 C with 5% CO2. Encapsulated cells were kept under a basal level of 5 x 10-4 IU hCG. The cells were stimulated with hCG (0.06 IU), sampled, and tested for testosterone following the same protocol used with the unencapsulated cells. The control experiments were performed as described for the unencapsulated cells. The viability of the encapsulated cells was measured using the MTT assay: 50 µl of MTT solution (5 mg/ml) was added to 0.5 ml of microspheres in medium and incubated at 37 C for 5 h. The medium was removed, and the microcapsules were incubated for 24 h with 1 ml of 0.4% HCl in isopropranolol. The OD was measured in a spectrophotometer using a wavelength of 570 nm.

Microencapsulated Leydig cell implantation
Five groups of 12 male Sprague Dawley rats, 56–70 d old, were castrated 1 wk before Leydig cell or unencapsulated cell administration. Approximately 5 x 106 Leydig cells were suspended in 0.5 ml of PBS and injected ip (group I) or sc (group II) into the castrated rats. The third and fourth groups received a suspension of 5 x 106 unencapsulated cells ip and sc, respectively. Group V rats served as controls and did not receive any treatment. As an additional control, testosterone levels were measured in six normal rats at the same time points as the castrated rats. In all experiments, serum samples were kept at -20 C until total testosterone levels were measured using RIAs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leydig-cell characterization
Approximately 70–90% of the cells retrieved from band III of the Percoll gradient were ß-HSD positive as measured by the 3ß-HSD detection assay (density of 1.070 g/ml) and by immunocytochemistry (Fig. 1Go). Approximately 55 x 106 Leydig cells were recovered from 20 rat testes. The MTT assay, together with the trypan blue exclusion test, showed that 95–100% of the cultured Leydig cells were viable for the duration of the experiment. Leydig cells at a density of 3 x 106 were cultured in each well of a 24-well plate. A minimum of five wells was used for each experiment, and each experiment was repeated at least three times.



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  		FIG. 1. Characterization of isolated Leydig cells. A, Positive staining for 3ß-HSD (magnification, x250); B, positive staining with anti-3ß-HSD antibody (magnification, x250).

 
Testosterone production by unencapsulated Leydig cells in vitro
In the first set of experiments, the medium from the cultured Leydig cells (37 C) was sampled before and 4, 8, and 24 h after hCG stimulation. Testosterone secretion was dramatically elevated after hCG stimulation compared with basal control (Fig. 2AGo). Testosterone was detected at all time points, with the highest levels at 24 h (149 ng/106 cells) post stimulation. Testosterone levels were below the sensitivity of the assay in samples taken from cultured Leydig cells that did not receive a basal level of hCG and were not stimulated with hCG.



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  		FIG. 2. The response of cultured rat Leydig cells to a maximum dose of hCG (0.04 IU) over a period of 24 h. The experiments were performed at 37 C (A) and at 32 C (B). The cells were maintained at a basal level of hCG before additional hCG stimulation.

 
The effect of temperature on the cultured Leydig cells and its effect on testosterone secretion were evaluated. Leydig cells were cultured at either 32 C or 37 C and were stimulated with hCG every 24 h. Leydig cells cultured at 32 C did not show an increased testosterone secretion when compared with Leydig cells cultured at 37 C (Fig. 2BGo).

Leydig cell microencapsulation
Based on the MM of testosterone (300 Da), PLL of 21-kDa MM and 1.2% sodium alginate with a high gluronic content (>65%) were chosen. PLL with a MM ranging between 16 and 22 kDa produced a semipermeable membrane with a MM cut off of 70 kDa, i.e. preventing the diffusion of cells and antibodies.

The microcapsules containing Leydig cells had a spherical shape with an average diameter of 0.7 mm ± 0.06 mm (Fig. 3Go). MTT assays performed daily on the microencapsulated cells showed that the cells remained viable during the experiments, but the overall viability was approximately 20% less than the unencapsulated cells.



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  		FIG. 3. Light microscopy of encapsulated Leydig cells (magnification, x25; 0.7 mm in diameter). The single cells were evenly distributed within the microcapsules.

 
In vitro secretion of testosterone from cultured microencapsulated Leydig cells
Preliminary studies in which different concentrations of hCG (0.02, 0.04, 0.06, and 0.08 IU) were used for Leydig cell stimulation demonstrated that a concentration of 0.06 IU was needed to achieve the highest levels of testosterone. A concentration of 0.08 IU did not have an additive effect. Therefore, an hCG concentration of 0.06 IU was used for all the microencapsulated cell experiments.

Testosterone secretion from encapsulated Leydig cells in response to hCG was highest 24 h after hCG stimulation. There was no significant difference in testosterone secretion when cells were cultured at either 32 C or 37 C (Fig. 4Go). Testosterone could not be detected in the medium of encapsulated Leydig cells that did not receive a basal level or stimulation with hCG.



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  		FIG. 4. The response of rat microencapsulated Leydig cells to a maximum dose of hCG (0.06 IU) over a period of 24 h. The experiments were performed at 37 C (A) and at 32 C (B). The microencapsulated cells were maintained at basal hCG levels before maximal dose stimulation with hCG. In the control experiments, cells were not stimulated with hCG.

 
In vivo secretion of testosterone from microencapsulated Leydig cells
Total testosterone levels measured in the serum of castrated rats that were injected with 5 x 106 encapsulated Leydig cells ip were between 0.23 and 0.51 ng/ml for more than 4 wk (Fig. 5AGo). These animals did not receive any exogenous hCG stimulation. Similar testosterone levels (0.24–0.48 ng/ml) were obtained when encapsulated Leydig cells were injected sc (Fig. 5BGo). However, testosterone was detected for a longer time period in the sc group (43 d) when compared with the ip group (35 d). After these time periods, testosterone levels were below the detection sensitivity of the assay. The average testosterone level of the uncastrated control rats was 1.7 ng/ml. The testosterone levels in the castrated control rats were below the detection sensitivity of the assay. The castrated rats that received unencapsulated Leydig cells showed no detection of testosterone.



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  		FIG. 5. Serum testosterone levels in castrated rats without Leydig cell implantation and in castrated rats that received 5 x 106 encapsulated Leydig cells ip (A) and sc (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Testosterone is essential for normal spermatogenesis and fertility and is produced in males mainly by the testicular Leydig cells. Alginate-PLL microspheres were chosen for Leydig cell encapsulation due to their biocompatibility and the mild preparation technique, which is performed entirely in aqueous conditions. Alginate-PLL capsules consist of the following three main components: a hydrogel core material of calcium alginate enclosed by a polyanion-polycation complex membrane and an outer coating of a polyanion. The internal hydrogel core entraps the cells rapidly while spherical beads are formed and serve as a binding template for the polycation, PLL. The semipermeable membrane formed from the interaction of alginate and PLL, with the proper MM, allows for the controlled delivery of bioactive substances in vitro and in vivo while preventing the diffusion of antibodies and other components of the immune system. An additional advantage of this system is the possible administration of the microspheres by injection, thus avoiding a surgical procedure.

The present study demonstrates that isolated Leydig cells that are encapsulated in alginate-PLL microspheres are able to deliver testosterone in vitro and in vivo. Encapsulated Leydig cells that were stimulated with hCG secreted high levels of testosterone in culture, but the testosterone levels were lower then those obtained from the unencapsulated cells. These differences may be due to cell death upon encapsulation. In addition, up to 20% of the cells can be lost during the process of encapsulation itself. Even though the encapsulated Leydig cells in vitro secreted lower levels of testosterone, they did so for up to 6 d, whereas the unencapsulated cells stopped producing testosterone by the third day.

The encapsulated and unencapsulated Leydig cells were found to be resistant to temperature changes, which supports previous reports (16). This finding broadens the possible in vivo sites for Leydig cell transplantation. The intraperitoneal cavity was the first site chosen for cell implantation due to its generous vascular and nutritional capabilities. Castrated rats that were administered encapsulated Leydig cells ip maintained a testosterone level between 0.23 and 0.51 ng/ml. These testosterone levels were lower than the ones detected in the control rats that were not castrated. Only 5 x 106 microencapsulated cells were implanted in each animal, which represents approximately only 10% of the normal adult rat Leydig cell population, yet this resulted in serum testosterone levels of up to 40% of normal. Similar levels of testosterone were obtained when the encapsulated Leydig cells were injected sc (0.28–0.48 ng/ml). Testosterone levels were detected up to 43 d post implantation. Clinically, testosterone is usually delivered for supplementation and not for full replacement therapy. Therefore, encapsulated Leydig cells may be potentially useful for clinical supplementation.

Other researchers have shown that implanted sc rat Leydig cells on a gelatin sponge are able to secrete testosterone but only for 1 wk and at low levels. These results were explained by a probable high degree of cell death upon implantation and fibroblast infiltration onto the gelatin sponge (7). Unencapsulated cells injected ip or sc failed to produce any testosterone in our study, even with hCG stimulation. However, the cells were injected without a carrier, which may explain the difference between our results and those described above. In addition, the cell encapsulation system used in our study would prevent the infiltration of other cells types, such as fibroblasts. A three-dimensional environment, which has been shown to increase the enzymatic and steroidogenic activities of cultured Leydig cells, is provided by the microencapsulation system (17).

This study shows that encapsulated Leydig cells are able to secrete testosterone both in vitro and in vivo. Stable levels of serum testosterone were achieved using this system.


   Footnotes
 
Abbreviations: hCG, Human chorionic gonadotropin; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; MM, molecular mass; MTT, 3-(4,5- dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; PLL, poly-L-lysine.

Received April 1, 2003.

Accepted for publication July 14, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bhasin S, Bremner WJ 1997 Clinical review 85: emerging issues in androgen replacement therapy. J Clin Endocrinol Metab 82:3–8[Free Full Text]
  2. Cantrill JA, Dewis P, Large DM, Newman M, Anderson DC 1984 Which testosterone replacement therapy? Clin Endocrinol (Oxf) 21:97–107[Medline]
  3. Snyder PJ, Lawrence DA 1980 Treatment of male hypogonadism with testosterone enanthate. J Clin Endocrinol Metab 51:1335–1339[Abstract/Free Full Text]
  4. McClellan KJ, Goa KL 1998 Transdermal testosterone. Drugs 55:253–258; discussion 259[CrossRef][Medline]
  5. Santen RJ, Swerdloff RS, Clinical aspects of androgen therapy. Workshop Conference on Androgen Therapy: Biologic and Clinical Consequences, Penn State Workshop Proceedings, Philadelphia, PA, 1990, p 40
  6. Tai J, Johnson HW, Tze WJ 1989 Successful transplantation of Leydig cells in castrated inbred rats. Transplantation 47:1087–1089[Medline]
  7. van Dam JH, Teerds KJ, Rommerts FF 1989 Transplantation and subsequent recovery of small amounts of isolated Leydig cells. Arch Androl 22:123–129[Medline]
  8. De Vos P, De Haan B, Van Schilfgaarde R 1997 Effect of the alginate composition on the biocompatibility of alginate-polylysine microcapsules. Biomaterials 18:273–278[CrossRef][Medline]
  9. Tai IT, Sun AM 1993 Microencapsulation of recombinant cells: a new delivery system for gene therapy. FASEB J 7:1061–1069[Abstract]
  10. Lim F, Sun AM 1980 Microencapsulated islets as bioartificial endocrine pancreas. Science 210:908–910[Abstract/Free Full Text]
  11. Lanza RP, Hayes JL, Chick WL 1996 Encapsulated cell technology. Nat Biotechnol 14:1107–1111[CrossRef][Medline]
  12. Joki T, Machluf M, Atala A, Zhu J, Seyfried NT, Dunn IF, Abe T, Carroll RS, Black PM 2001 Continuous release of endostatin from microencapsulated engineered cells for tumor therapy. Nat Biotechnol 19:35–39[CrossRef][Medline]
  13. Schuch G, Machluf M, Bartsch Jr G, Nomi M, Richard H, Atala A, Soker S 2002 In vivo administration of vascular endothelial growth factor (VEGF) and its antagonist, soluble neuropilin-1, predicts a role of VEGF in the progression of acute myeloid leukemia in vivo. Blood 100:4622–4628[Abstract/Free Full Text]
  14. Lee MS, Hong SJ, Choi HR, Rha KH 1994 Testosterone productivity and histostructural changes of autotransplanted rat Leydig cells. Yonsei Med J 35:260–270[Medline]
  15. Machluf M, Regev O, Peled Y, Kost J, Cohen S 1997 Characterization of microencapsulated liposome systems for the controlled delivery of liposome-associated macromolecules. J Control Release 43:35–45[CrossRef]
  16. Sharpe RM, Maddocks S, Kerr JB 1990 Cell-cell interactions in the control of spermatogenesis as studied using Leydig cell destruction and testosterone replacement. Am J Anat 188:3–20[CrossRef][Medline]
  17. Kmicikiewicz I, Koziel E, Pelczar U, Pierscinski A, Bilinska B 1998 Mouse Leydig cell culture on microcarriers. Acta Histochem 100:297–307[Medline]



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