Lacrimal gland primary acinar cell culture: the role of insulin

The goal of the present study was to establish a protocol for primary culture of lacrimal gland acinar cells (LGACs) and to assess the effect of adding insulin to the culture media.

Methods:

LGACs were isolated and cultured from lacrimal glands of Wistar male rats. The study outcomes included cell number, viability, and peroxidase release over time and in response to three concentrations of insulin (0.5, 5.0, and 50.0 μg/mL).

Results:

In LGAC primary culture, cells started to form clusters by day 3. There was a time-response pattern of peroxidase release, which rose by day 6, in response to carbachol. Culture viability lasted for 12 days. An insulin concentration of 5.0 μg/mL in the culture medium resulted in higher viability and secretory capacity.

Conclusions:

The present method simplifies the isolation and culture of LGACs. The data confirmed the relevance of adding insulin to maintain LGACs in culture.

Keywords:
Acinar cells; Lacrimal gland; Lacrimal apparatus; Insulin; Peroxidase; Cell Count; Regenerative medicine; Tissue engineering; Animals; Rats, Wistar

Authorship

Leonardo Tannus Malki

Department of Ophthalmology, Otorhinolaryngology and Head & Neck Surgery. Faculdade de Medicina de Ribeirão Preto (FMRP), Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil.Universidade de São PauloBrazilRibeirão Preto, SP, BrazilDepartment of Ophthalmology, Otorhinolaryngology and Head & Neck Surgery. Faculdade de Medicina de Ribeirão Preto (FMRP), Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil.

Ana Carolina Dias

Angelica Gobbi Jorge

Carolina Maria Módulo

Eduardo Melani Rocha Corresponding author: Eduardo Melani Rocha. Department of Ophthalmology. Otorhinolaryngology and Head & Neck Surgery. Faculdade de Medicina de Ribeirão Preto - Universidade de São Paulo - Av. Bandeirantes, 3.900 - Ribeirão Preto, SP - 14049-900 - Brazil - E-mail: emrocha@fmrp.usp.br

Corresponding author: Eduardo Melani Rocha. Department of Ophthalmology. Otorhinolaryngology and Head & Neck Surgery. Faculdade de Medicina de Ribeirão Preto - Universidade de São Paulo - Av. Bandeirantes, 3.900 - Ribeirão Preto, SP - 14049-900 - Brazil - E-mail: emrocha@fmrp.usp.br

Disclosure of potential conflicts of interest: None of the authors have any potential conflict of interest to disclose.

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (8)
Tables (1)

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Figure 1
Schematic study design showing the steps involved in and timing of the evaluation of LGAC growth in primary culture.

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Figure 2
Illustrative photos of the key steps involved in LGAC primary culture. A) Rat exorbital lacrimal gland isolated (arrow). B) Lacrimal gland cutting in a Petri dish and embedding of small fragments in culture medium. C) Enzymatic digestion of the lacrimal gland on a magnetic stirrer using a spinner flask. D) Lacrimal gland cell pellet after centrifugation (arrow). E) LGAC counting and viability testing using a Neubauer chamber and trypan blue after isolation.

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Figure 3
A) A low amount of aquaporin-5-positive LGACs (3%) was observed in the supernatant. B) A higher amount of positive cells (20%) was observed in the pellet (arrow), indicating the efficiency of the isolation method.

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Figure 4
Counts of LG acinar cells in primary culture on four consecutive days, using supplemented low-glucose DMEM. Comparative analysis of four substrates: plastic, poly-L-lysine, fibronectin, and Matrigel at day 0 (black columns) and day 4 (white columns) (n=3 wells/condition).

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Figure 5
Microscopic aspects of LG acinar cells in culture on day 4. A) The wave front of cell proliferation by the third day (100x magnification). B) LG cells forming clusters similar to acinar units (arrow) by the third day (200x magnification). C) Structures compatible with secretory granules in the cytoplasm (arrow) were observed on the fifth day (200x magnification). D) Different morphologic types of LG cells, suggesting the proliferation of remaining myoepithelial and mesenchymal cells by the third week (400x magnification).

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Figure 6
Counts of LG acinar cells in primary culture over a span of 12 days, using supplemented low-glucose DMEM and Matrigel substrate; comparative analysis of cell number (A) and viability (B) every 3 days, from the seeding day (0) until day 12 (n=3 wells/condition).

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Figure 7
Peroxidase activity in the culture medium using Matrigel substrate of LG acinar cells in primary culture from day 0 to 12. A) The culture medium containing the standard insulin concentration (5.0 μg/mL) was collected at the indicated days, prior to and 30 min after incubation with 100 mM carbachol. Peroxidase activity was analyzed as described in the methods (n = 2-4 samples/condition). B) The effect of insulin concentration on secretory activity over a period of 12 days in response to 100 mM carbachol stimulation. LGACs were cultured using three concentrations of insulin (0.5, 5.0, and 50.0 μg/mL). The culture media was collected on the indicated days (days 0-12) and peroxidase activity was measured (n=3/condition in duplicates) as described in the methods section (black filled bars, 5.0 μg/mL insulin; white filled bars, 0.5 μg/mL insulin; grey filled bars, 50.0 μg/mL of insulin).

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Figure 8
The effect of insulin concentration in the supplemented low-glucose DMEM media on LGAC proliferation in primary culture, using Matrigel substrate over a span of 12 days. Comparative analysis of cell number (A) and viability (B) in response to three concentrations of insulin in the medium (0.5, 5.0, and 50.0 μg/mL) was evaluated. The culture was discontinued at the indicated days (0 through 12), and cell counting and viability testing was performed (n=3/condition) using a Neubauer chamber and trypan blue staining (black filled bars, 5.0 μg/mL insulin; white filled bars, 0.5 μg/mL insulin; grey filled bars, 50.0 μg/mL of insulin).

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Table 1
Composition of the supplemented low-glucose DMEM used in the primary culture of LG acinar cells

Figure 1 Schematic study design showing the steps involved in and timing of the evaluation of LGAC growth in primary culture.

Figure 2 Illustrative photos of the key steps involved in LGAC primary culture. A) Rat exorbital lacrimal gland isolated (arrow). B) Lacrimal gland cutting in a Petri dish and embedding of small fragments in culture medium. C) Enzymatic digestion of the lacrimal gland on a magnetic stirrer using a spinner flask. D) Lacrimal gland cell pellet after centrifugation (arrow). E) LGAC counting and viability testing using a Neubauer chamber and trypan blue after isolation.

Figure 3 A) A low amount of aquaporin-5-positive LGACs (3%) was observed in the supernatant. B) A higher amount of positive cells (20%) was observed in the pellet (arrow), indicating the efficiency of the isolation method.

Figure 4 Counts of LG acinar cells in primary culture on four consecutive days, using supplemented low-glucose DMEM. Comparative analysis of four substrates: plastic, poly-L-lysine, fibronectin, and Matrigel at day 0 (black columns) and day 4 (white columns) (n=3 wells/condition).

Figure 5 Microscopic aspects of LG acinar cells in culture on day 4. A) The wave front of cell proliferation by the third day (100x magnification). B) LG cells forming clusters similar to acinar units (arrow) by the third day (200x magnification). C) Structures compatible with secretory granules in the cytoplasm (arrow) were observed on the fifth day (200x magnification). D) Different morphologic types of LG cells, suggesting the proliferation of remaining myoepithelial and mesenchymal cells by the third week (400x magnification).

Figure 6 Counts of LG acinar cells in primary culture over a span of 12 days, using supplemented low-glucose DMEM and Matrigel substrate; comparative analysis of cell number (A) and viability (B) every 3 days, from the seeding day (0) until day 12 (n=3 wells/condition).

Figure 7 Peroxidase activity in the culture medium using Matrigel substrate of LG acinar cells in primary culture from day 0 to 12. A) The culture medium containing the standard insulin concentration (5.0 μg/mL) was collected at the indicated days, prior to and 30 min after incubation with 100 mM carbachol. Peroxidase activity was analyzed as described in the methods (n = 2-4 samples/condition). B) The effect of insulin concentration on secretory activity over a period of 12 days in response to 100 mM carbachol stimulation. LGACs were cultured using three concentrations of insulin (0.5, 5.0, and 50.0 μg/mL). The culture media was collected on the indicated days (days 0-12) and peroxidase activity was measured (n=3/condition in duplicates) as described in the methods section (black filled bars, 5.0 μg/mL insulin; white filled bars, 0.5 μg/mL insulin; grey filled bars, 50.0 μg/mL of insulin).

Figure 8 The effect of insulin concentration in the supplemented low-glucose DMEM media on LGAC proliferation in primary culture, using Matrigel substrate over a span of 12 days. Comparative analysis of cell number (A) and viability (B) in response to three concentrations of insulin in the medium (0.5, 5.0, and 50.0 μg/mL) was evaluated. The culture was discontinued at the indicated days (0 through 12), and cell counting and viability testing was performed (n=3/condition) using a Neubauer chamber and trypan blue staining (black filled bars, 5.0 μg/mL insulin; white filled bars, 0.5 μg/mL insulin; grey filled bars, 50.0 μg/mL of insulin).

Table 1 Composition of the supplemented low-glucose DMEM used in the primary culture of LG acinar cells

Suplement	Final concentration
Putrescin	1.0 mM
Reduced glutathione	10.0 μg/ML
Ascorbic acid	50.0 μg/ML
Dexamethasone	10.0 μg/ML
Insulin	5.0 μg/ML
Selenium	5.0 μg/ML
Transferrin	5.0 μg/ML

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Lacrimal gland primary acinar cell culture: the role of insulin

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[1] Corresponding author: Eduardo Melani Rocha. Department of Ophthalmology. Otorhinolaryngology and Head & Neck Surgery. Faculdade de Medicina de Ribeirão Preto - Universidade de São Paulo - Av. Bandeirantes, 3.900 - Ribeirão Preto, SP - 14049-900 - Brazil - E-mail: emrocha@fmrp.usp.br