Perceived Conjunctival Foreign Material Egress in Morgellons Disease: A Case Study.

The purpose of this report is to present a case of a 63-year-old man with orbital Morgellons disease. A 63-year-old man presented reporting 15 years of daily egress of different foreign bodies apparently found in the superior fornices of both eyes, exhibiting a classic manifestation known as the matchbox sign. He described the symptoms starting after a facial trauma. The patient stated that at several points over the 15-year course of his condition, he was so distressed that he had contemplated suicide. On multiple exams by a range of ophthalmic professionals, there was no evidence of foreign body. Further investigation involving MRI and plain radiographs demonstrated similar lack of findings. A trial of gabapentin was performed without improvement in symptoms. He discontinued care 5 months later. Morgellons disease is a poorly understood condition, particularly ophthalmic presentations of the disease. Despite extensive investigation, the exact cause of Morgellons disease remains unclear, and there is no definitive treatment for the condition. We highlight the importance of empathetic listening in building trust, as a means of helping the patient to seek psychological help.

Orbital indeterminate cell histiocytosis.

An 8-year-old female presented to the oculoplastics clinic with 3 months of left upper eyelid fullness and edema. Examination showed a mass in the left anterior superior orbit with erythema. Imaging demonstrated a well-circumscribed superolateral orbital mass that was T1 hypointense and T2 hypo-to-iso intense with contrast enhancement. An incisional biopsy was performed via an upper lid crease incision. Histopathology showed aggregates of histiocytic cells with fibrosis and infiltration of eosinophils. Immunohistochemistry revealed positive CD68 and CD163 staining and negative langerin staining, confirming the diagnosis of indeterminate cell histiocytosis. There was no systemic involvement or associated dermatologic findings. Repeat exam 3 months later showed no change in the size of the lesion and the patient was referred to hematology-oncology for treatment. On most recent exam, the patient had no new symptoms or side effects following 3 months of oral hydroxyurea (25 mg/kg/day). Repeat orbital imaging showed no progression of the lesion and the patient will be monitored closely. Here, we report a rare case of isolated orbital indeterminate cell histiocytosis in a young child.

Teprotumumab-Related Adverse Events in Thyroid Eye Disease: A Multicenter Study.

PURPOSE: To assess the duration, incidence, reversibility, and severity of adverse events (AEs) in patients with thyroid eye disease (TED) treated with teprotumumab. DESIGN: Multicenter, retrospective, observational cohort study. PARTICIPANTS: Patients with TED of all stages and activity levels treated with at least 4 infusions of teprotumumab. METHODS: Patients were treated with teprotumumab between February 2020 and October 2022 at 6 tertiary centers. Adverse event metrics were recorded at each visit. MAIN OUTCOME MEASURES: The primary outcomes measure was AE incidence and onset. Secondary outcome measures included AE severity, AE reversibility, AE duration, proptosis response, clinical activity score (CAS) reduction, and Gorman diplopia score improvement. RESULTS: The study evaluated 131 patients. Proptosis improved by 2 mm or more in 77% of patients (101/131), with average proptosis improvement of 3.0 ± 2.1 mm and average CAS reduction of 3.2 points. Gorman diplopia score improved by at least 1 point for 50% of patients (36/72) with baseline diplopia. Adverse events occurred in 81.7% of patients (107/131). Patients experienced a median of 4 AEs. Most AEs were mild (74.0% [97/131]), 28.2% (37/131) were moderate, and 8.4% (11/131) were severe. Mean interval AE onset was 7.9 weeks after the first infusion. Mean resolved AE duration was 17.6 weeks. Forty-six percent of patients (60/131) demonstrated at least 1 persistent AE at last follow-up. Mean follow-up was 70.2 ± 38.5 weeks after the first infusion. The most common type of AEs was musculoskeletal (58.0% [76/131]), followed by gastrointestinal (38.2% [50/131]), skin (38.2% [50/131]), ear and labyrinth (30.5% [40/131]), nervous system (20.6% [27/131]), metabolic (15.3% [20/131]), and reproductive system (12.2% [16/131]). Sixteen patients (12.2%) discontinued therapy because of AEs, including hearing loss (n = 4), inflammatory bowel disease flare (n = 2), hyperglycemia (n = 1), muscle spasms (n = 1), and multiple AEs (n = 8). CONCLUSIONS: Adverse events are commonly reported while receiving teprotumumab treatment. Most are mild and reversible; however, serious AEs can occur and may warrant treatment cessation. Treating physicians should inform patients about AE risk, properly screen patients before treatment, monitor patients closely throughout therapy, and understand how to manage AEs should they develop. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Teprotumumab for the Treatment of Recalcitrant Thyroid Eye Disease.

PURPOSE: Teprotumumab, an insulin-like growth factor 1 receptor monoclonal antibody, is FDA-approved to treat thyroid eye disease (TED). The initial clinical trials excluded patients with previous orbital irradiation, surgery, glucocorticoid use (cumulative dose >1 gm), or prior biologic treatment. Information on the use of teprotumumab for patients who failed prior therapy is limited. Our purpose is to characterize the efficacy of teprotumumab for the treatment of recalcitrant TED. METHODS: This is a multicenter retrospective study of all patients treated with teprotumumab for moderate-to-severe TED after failing conventional therapy with corticosteroids, orbital radiation, surgical decompression, biologics, or other steroid-sparing medications. Treatment failure was defined as an incomplete response to or reactivation after previous treatment. Only patients who received at least 4 infusions of teprotumumab were included in the analysis. Primary outcome measures comprised proptosis response (≥2 mm reduction in the study eye without a similar increase in the other eye), clinical activity score (CAS) response (≥2-point reduction in CAS), and diplopia response (≥1 point improvement in Gorman diplopia score in patients with baseline diplopia) following treatment. Adverse events and risk factors for recalcitrant disease were also evaluated. RESULTS: Sixty-six patients were included in this study, 46 females and 20 males. Average age was 59.3 years (range 29-93). The mean duration of disease from TED diagnosis to first infusion was 57.8 months. The proptosis, CAS, and diplopia responses in this recalcitrant patient population were 85.9%, 93.8%, and 69.1%, respectively. Patients experienced a mean reduction in proptosis of 3.1 ± 2.4 mm and a mean improvement in CAS of 3.8 ± 1.6. Patients who underwent prior decompression surgery experienced a statistically significant decrease in diplopia response (46.7% vs. 77.5%, p = 0.014) and proptosis response (75.0% vs. 90.9%, p = 0.045) when compared with nondecompression patients. Additionally, there were no significant differences in proptosis, CAS, and diplopia responses between patients with acute (defined as disease duration <1 year) versus chronic (disease duration ≥1 year) TED. While most adverse events were mild to moderate, 4 patients reported serious adverse events related to persistent hearing loss. CONCLUSIONS: Patients with recalcitrant TED demonstrated a significant improvement after teprotumumab in each of the primary study outcomes. The degree of proptosis reduction, diplopia response, and CAS improvement in the recalcitrant group were similar to those of treatment-naïve patients from the pivotal clinical trials. Patients with a prior history of orbital decompression, however, demonstrated poor improvement in diplopia and less reduction in proptosis than surgery naïve patients. These results indicate that teprotumumab is a treatment option for the treatment of patients with TED recalcitrant to prior medical therapies.

Teprotumumab and Orbital Decompression for the Management of Proptosis in Patients With Thyroid Eye Disease.

PURPOSE: To compare outcomes of patients with thyroid eye disease treated with teprotumumab or orbital decompression, or both in sequence. METHODS: Patients with thyroid eye disease and treated with decompression, teprotumumab, or both were included. Four groups were defined: decompression only, teprotumumab only, teprotumumab first with decompression later, and decompression first with teprotumumab later. The primary outcome was change in exophthalmometry. Secondary outcomes included change in extraocular muscle motility, strabismus, diplopia, and side effects. RESULTS: One hundred and thirty-nine patients were included. The mean duration for early follow-up was 1.2 months for both decompression and teprotumumab groups. The mean late follow-up was 14.4 and 8.2 months for the decompression and teprotumumab groups respectively. Mean change in exophthalmometry was significantly greater for the decompression group (3.5 mm) compared with teprotumumab (2.0 mm) at late follow-up. Improvement in total extraocular muscle restriction was significantly greater in the teprotumumab group (14.7 degrees) than in the decompression group (2.6 degrees). The teprotumumab group had a significantly higher percentage of patients with diplopia score >1 at baseline and late follow-up (p < 0.01) compared with the decompression group. Additional treatment with teprotumumab or decompression when previously treated with the opposite had similar proptosis reduction effect as that therapy alone. CONCLUSIONS: Surgical decompression has a greater proptosis reduction effect than teprotumumab, whereas teprotumumab better improves extraocular muscle motility. The addition of teprotumumab or decompression to a previous course of the opposite adds a similar effect to the supplemental treatment alone.

Moral Injury: How It Affects Us and Tools to Combat It.

Introduction: Moral injury comprises feelings of guilt, despair, shame, and/or helplessness from having one's morals transgressed. Those underrepresented in health care are more likely to experience moral injury arising from micro- and macroaggressions. This workshop was designed for interprofessional health care providers ranging from students to program leadership to raise awareness about moral injury and provide tools to combat it. Methods: This 75-minute interactive workshop explored moral injury through a health care lens. It included components of lecture, case-based learning, small-group discussion, and individual reflection. Participants completed anonymous postworkshop evaluations, providing data on satisfaction and intention to change practice. We used descriptive statistics to analyze the quantitative data and applied content analysis to the qualitative data. Results: The workshop was presented at two local academic conferences. Data were collected from 34 out of 60 participants, for a response rate of 57%. Ninety-seven percent of participants felt the workshop helped them define and identify moral injury and was a valuable use of their time, as well as indicating they would apply the information learned in their daily life. One hundred percent would recommend the workshop to a friend or colleague. Almost half felt they could implement strategies to address moral injury after participating in the workshop. Discussion: This workshop proved to be a valuable tool to define and discuss moral injury. The materials can be adapted to a broad audience.

Outcomes of Patients With Thyroid Eye Disease Partially Treated With Teprotumumab.

PURPOSE: In response to the coronavirus (COVID-19) pandemic, teprotumumab production was temporarily halted with resources diverted toward vaccine production. Many patients who initiated treatment with teprotumumab for thyroid eye disease were forced to deviate from the standard protocol. This study investigates the response of teprotumumab when patients receive fewer than the standard 8-dose regimen. METHODS: This observational cross-sectional cohort study included patients from 15 institutions with active or minimal to no clinical activity thyroid eye disease treated with the standard teprotumumab infusion protocol. Patients were included if they had completed at least 1 teprotumumab infusion and had not yet completed all 8 planned infusions. Data were collected before teprotumumab initiation, within 3 weeks of last dose before interruption, and at the visit before teprotumumab reinitiation. The primary outcome measure was reduction in proptosis more than 2 mm. Secondary outcome measures included change in clinical activity score (CAS), extraocular motility restriction, margin reflex distance-1 (MRD1), and reported adverse events. RESULTS: The study included 74 patients. Mean age was 57.8 years, and 77% were female. There were 62 active and 12 minimal to no clinical activity patients. Patients completed an average of 4.2 teprotumumab infusions before interruption. A significant mean reduction in proptosis (-2.9 mm in active and -2.8 mm in minimal to no clinical activity patients, P < 0.01) was noted and maintained during interruption. For active patients, a 3.4-point reduction in CAS ( P < 0.01) and reduction in ocular motility restriction ( P < 0.01) were maintained during interruption. CONCLUSIONS: Patients partially treated with teprotumumab achieve significant reduction in proptosis, CAS, and extraocular muscle restriction and maintain these improvements through the period of interruption.

Advances in the Treatment of Thyroid Eye Disease Associated Extraocular Muscle Myopathy and Optic Neuropathy.

PURPOSE OF REVIEW: To review emerging treatments for thyroid eye disease (TED) associated extraocular muscle myopathy and dysthyroid optic neuropathy (DON). RECENT FINDINGS: Emerging targeted biologic therapies may alter the disease course in TED. Teprotumumab, a type I insulin-like growth factor receptor inhibitor, is the most recent addition to the treatments available for TED-associated extraocular muscle myopathy causing diplopia. Small studies also suggest a potential therapeutic benefit for DON. Various recent studies have also expanded our knowledge on conventional TED therapies. The therapeutic landscape of TED and its sequelae has evolved in recent years. New targeted therapies have the potential to reduce the extraocular muscle and orbital volume expansion which can lead to diplopia and vision loss from optic nerve compression. Longer term efficacy and durability data is needed to determine the role biologics, such as teprotumumab, should play in the treatment of TED patients compared to the current standard of care.

Hearing Dysfunction After Treatment With Teprotumumab for Thyroid Eye Disease.

PURPOSE: To characterize the frequency, severity, and resolution of hearing dysfunction in patients treated with teprotumumab for thyroid eye disease (TED). DESIGN: Prospective observational case series. METHODS: Ophthalmic examination and adverse event assessment, including otologic symptoms, were performed at baseline, after infusions 2, 4, and 8, and at 6-month follow-up in consecutive patients who received at least 4 teprotumumab infusions. Laboratory test results were collected at baseline and during treatment. Audiometry, patulous eustachian tube (PET) testing, and otolaryngology evaluation were obtained for patients with new or worsening otologic symptoms, with a subset obtaining baseline and posttreatment testing. RESULTS: Twenty-seven patients were analyzed (24 females, 3 males, average 56.3 years old). Twenty-two patients (81.5%) developed new subjective otologic symptoms, after a mean of 3.8 infusions (SD 1.8). At 39.2-week average follow-up after the last infusion, most patients with tinnitus (100%), ear plugging/fullness (90.9%), and autophony (83.3%) experienced symptom resolution, whereas only 45.5% (5 of 11) of patients with subjective hearing loss/decreased word comprehension experienced resolution. Six patients underwent baseline and posttreatment audiometry, 5 of whom developed teprotumumab-related sensorineural hearing loss (SNHL) and 1 patient also developed PET. Three of the 5 patients with teprotumumab-related SNHL had persistent subjective hearing loss at last follow-up. A prior history of hearing loss was discovered as a risk factor for teprotumumab-related SNHL (P = .008). CONCLUSIONS: Hearing loss is a concerning adverse event of teprotumumab, and its mechanism and reversibility should be further studied. Until risk factors for hearing loss are better understood, we recommend baseline audiometry with PET testing and repeat testing if new otologic symptoms develop. Screening, monitoring, and prevention guidelines are needed.

Trends in Leadership and Award Recognition Among Women in the American Society of Retina Specialists.

Purpose: This work evaluates trends in achievement of women in the retina field, through an analysis of gender representation in the American Society of Retina Specialists (ASRS). Methods: This retrospective, longitudinal study spans 1983 to 2020. Historical data classified by male or female gender were collected from ASRS's overall membership, board of directors and officers, and recipients of the 4 society awards. The proportion of each benchmark held by women was compared with prior decades since the founding of ASRS using the Fisher's exact test. Results: Women's representation increased from 11% of ASRS members in 2007 to 19.7% in 2020. From 2010 to 2019, women received a higher proportion of society awards (21.1%) compared with membership prior to the start of that decade. In 2020, women were proportionally well represented in board of director positions (21.9%) and held a significantly higher proportion of board positions than in the period 1983 to 1989 (P = .02). From 1983 to 2020, women held 4.3% (1 of 23) of presidencies. Conclusions: Although the number of women in retina is increasing, women remain underrepresented in the leadership of ASRS. Interventions to increase exposure to female mentorship and improve childcare benefits are warranted to engage female ophthalmology trainees in retina and ultimately society leadership.

Microphthalmia and orbital cysts in DiGeorge syndrome.

We report the case of a 4-month-old boy diagnosed with DiGeorge syndrome with novel ocular features. The patient was diagnosed through genetic testing, with a noted 22q11.2 deletion, and had the additional clinical findings of cardiac anomalies, Hirschsprung's disease, and intracranial microhemorrhages. Eye findings included bilateral microphthalmia, persistent fetal vasculature, chorioretinal coloboma, and a unilateral orbital cyst. Given no known additional inciting exposures, a dysgenic mechanism resulting in failed closure of developmental fissures associated with the chromosomal deletion likely gave rise to these combined pathologies.

Early efficacy of teprotumumab for the treatment of dysthyroid optic neuropathy: A multicenter study.

PURPOSE: To study post-interventional findings in patients with dysthyroid optic neuropathy (DON) treated with teprotumumab. OBSERVATIONS: In this multicenter observational Case series, patients with DON were treated with teprotumumab, an insulin-like growth factor I receptor inhibitor (10 mg/kg for the first infusion then 20 mg/kg for subsequent infusions, every three weeks for a total 8 infusions). This study included patients with acute and chronic thyroid eye disease (TED) with DON who had failed conventional therapies and were not candidates for surgical decompression. Data collected included best corrected visual acuity (BCVA), color vision, RAPD when present, and orbital CT or MRI. Proptosis, clinical activity score (CAS), Gorman diplopia score (GDS), and Humphrey visual fields (HVF) were also evaluated.Ten patients (6 women, 4 men) with an average age 64 years old were included in this study. Mean follow up after completion of infusions was 15 weeks. Baseline visual acuity (VA) impairment ranged from hand motion (HM) to 20/25 in affected eyes. All patients had pre-treatment orbital CT or MRI that confirmed orbital apex compression. Seventy percent of patients had objective improvement in DON after 2 infusions of teprotumumab measured as significant improvement in visual acuity, resolution of RAPD, or both. After completion of treatment, affected eyes had a mean BCVA improvement of 0.87 logMAR (p=0.0207), proptosis reduction of 4.7 mm (p<0.00001), CAS improvement of 5.25 points (p<0.00001), and GDS improvement of 0.75 points (p=0.160). All 6 patients who presented with an RAPD had resolution or improvement of RAPD. All 7 patients who presented with color vision deficits had normalization or improvement of color vision. CONCLUSIONS AND IMPORTANCE: Teprotumumab infusions resulted in medical decompression and objective resolution or improvement of dysthyroid optic neuropathy. Most patients had rapid improvement of visual acuity and reversal of RAPD. Post-infusion imaging demonstrated reduction in extraocular muscle size that correlated with improvement in visual dysfunction. However, patients who presented with longstanding severe visual loss had limited improvement. There was no recurrence of DON after completion of teprotumumab in our cohort.

Multi-compartment skull base orbital cavernous venous malformation: A rare presentation of a common orbital mass.

PURPOSE: We present a unique case of an orbital intraconal cavernous venous malformation that extended along the trigeminal nerve to the pterygopalatine and middle cranial fossa. Our aim is to describe an atypical presentation of this common orbital vascular mass. OBSERVATIONS: A 57-year-old female presented with right eye proptosis. Orbital magnetic resonance imaging demonstrated a lobulated contrast-enhancing mass involving the right intraconal orbital space, pterygopalatine fossa, and right middle cranial fossa, radiographically presumed to be a schwannoma. Intraoperative and histopathologic evaluation confirmed a cavernous venous malformation that extended along the trigeminal nerve. The mass, including its attachments to the cranial nerves and dura, was successfully removed via a combined transorbital and endoscopic endonasal approach. The patient recovered well with 20/20 vision, full extraocular movements, and resolution of proptosis. CONCLUSIONS: This a rare presentation of an orbital cavernous venous malformation not previously described. Cavernous venous malformations typically present as ovoid well-circumscribed lesions; however, they can also extend outside the orbit along the path of cranial nerves, as was observed in this case. These types of lesions should be included in the differential diagnosis of masses arising from or extending along cranial nerves, even when involving the orbit.

Teprotumumab for Dysthyroid Optic Neuropathy: Early Response to Therapy.

A 45-year-old male presented with active progressive thyroid eye disease refractory to intravenous steroids and right orbital radiation. Visual acuity, left relative afferent pupillary defect, and Humphrey visual field defects were consistent with worsening left dysthyroid optic neuropathy. Orbital MRI demonstrated extraocular muscle enlargement and effacement of the left optic nerve sheath. After 2 infusions of teprotumumab, the patient's visual acuity, relative afferent pupillary defect, Humphrey visual fields, proptosis, and extraocular muscle size improved. This is the first report of dysthyroid optic neuropathy responsive to teprotumumab, and it supports the need for further studies to better understand the role of teprotumumab in treating sight-threatening thyroid eye disease.

Quantitative Assessment of the Severity of Diabetic Retinopathy.

PURPOSE: To determine whether a quantitative approach to assessment of the severity of diabetic retinopathy (DR) lesions on ultrawide field (UWF) images can provide new parameters to predict progression to proliferative diabetic retinopathy (PDR). METHODS: One hundred forty six eyes from 73 participants with DR and 4 years of follow-up data were included in this post hoc analysis, which was based on a cohort of 100 diabetic patients enrolled in a previously published prospective, comparative study of UWF imaging at the Joslin Diabetes Center. Diabetic Retinopathy Severity Score level was determined at baseline and 4-year follow-up visits using mydriatic 7-standard field Early Treatment Diabetic Retinopathy Study (ETDRS) photographs. All individual DR lesions (hemorrhage [H], microaneurysm [ma], cotton wool spot [CWS], intraretinal microvascular abnormality [IRMA]) were manually segmented on stereographic projected UWF. For each lesion type, the frequency/number, surface area, and distances from the optic nerve head (ONH) were computed. These quantitative parameters were compared between eyes that progressed to PDR in 4 years and eyes that did not progress. Univariable and multivariable logistic regression analyses were performed to identify parameters that were associated with an increased risk for progression to PDR. RESULTS: A total of 146 eyes of 73 subjects were included in the final analysis. The mean age of the study cohort was 53.1 years, and 42 (56.8%) subjects were female. The number and surface area of H/ma's and CWSs were significantly (P ≤ .05) higher in eyes that progressed to PDR compared with eyes that did not progress by 4 years. Similarly, H/ma's and CWSs were located further away from the ONH (ie, more peripheral) in eyes that progressed (P < .05). DR lesion parameters that conferred a statistically significant increased risk for proliferative diabetic retinopathy in the multivariate model included hemorrhage area (odds ratio [OR], 2.63; 95% confidence interval [CI], 1.25-5.53), and greater distance of hemorrhages from the ONH (OR, 1.24; 95% CI, 0.97-1.59). CONCLUSIONS: Quantitative analysis of DR lesions on UWF images identifies new risk parameters for progression to PDR including the surface area of hemorrhages and the distance of hemorrhages from the ONH. Although these risk factors will need to be confirmed in larger, prospective studies, they highlight the potential for quantitative lesion analysis to inform the design of a more precise and complete staging system for diabetic retinopathy severity in the future. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Comparison of Subjective Assessment and Precise Quantitative Assessment of Lesion Distribution in Diabetic Retinopathy.

Importance: Predominantly peripheral disease in eyes with nonproliferative diabetic retinopathy (DR) is suggested as a potential strong risk factor for progression to proliferative disease. However, the reliability and optimal method for the assessment of lesion distribution are still uncertain. Objective: To compare agreement between subjective assessment and precise quantification of lesion burden in ultrawidefield (UWF) images of eyes with DR. Design, Setting, and Participants: This multisite cross-sectional study examines UWF pseudocolor images acquired from DR screening clinic patients from December 20, 2014, through August 1, 2014. Of 104 cases, 161 eyes with DR were included. Data analysis was conducted from June 1, 2016, through December 1, 2016 at the Doheny Image Reading Center. Main Outcomes and Measures: Distribution of DR lesions in eyes was assessed subjectively and quantitatively, and eyes were classified as having predominantly central lesions (PCLs) or predominantly peripheral lesions (PPLs). The frequency and surface area (SA) of each lesion type were quantified. Intergrader and subjective vs quantitative classification were compared for level of agreement. Several methods of determining PPL distribution were also compared. Results: On subjective frequency-based evaluation by graders, 133 eyes were classified as having PCL, and 28 eyes as having PPL. On exact quantification of lesion SA, 121 eyes were classified as PCL, and 40 eyes as having PPL. On SA-based quantification, 134 eyes were classified as having PCL, and 27 eyes as having PPL. There was a significant difference between qualitative and quantitative classification of DR lesion distribution for both frequency-based (mean difference [SD]: PCL, 6 [2]; PPL, 13 [6]; P < .001) and SA-based (mean difference [SD]: PCL, 6 [1]; PPL, 20 [7]; P < .001) methods. Both intergrader reproducibility and subjective vs quantitative agreement were higher with frequency-based classification. Conclusions and Relevance: Subjective assessment of PPL DR lesions on UWF images differed in some cases from precise quantitative assessments, particularly when considering the area of lesions. These findings highlight the benefit of objective quantitative approaches to DR assessment, which may facilitate the development of a more precise DR scoring system.

Type I collagen as an extracellular matrix for the in vitro growth of human small intestinal epithelium.

BACKGROUND: We previously reported in vitro maintenance and proliferation of human small intestinal epithelium using Matrigel, a proprietary basement membrane product. There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells. METHODS: Human small intestine was procured from fresh surgical pathology specimens. Small intestinal crypts were isolated using EDTA chelation. Intestinal subepithelial myofibroblasts were isolated from a pediatric sample and expanded in vitro. After suspension in Matrigel or type I collagen gel, crypts were co-cultured above a confluent layer of myofibroblasts. Crypts were also grown in monoculture with exposure to myofibroblast conditioned media; these were subsequently sub-cultured in vitro and expanded with a 1∶2 split ratio. Cultures were assessed with light microscopy, RT-PCR, histology, and immunohistochemistry. RESULTS: Collagen supported viable human epithelium in vitro for at least one month in primary culture. Sub-cultured epithelium expanded through 12 passages over 60 days. Histologic sections revealed polarized columnar cells, with apical brush borders and basolaterally located nuclei. Collagen-based cultures gave rise to monolayer epithelial sheets at the gel-liquid interface, which were not observed with Matrigel. Immunohistochemical staining identified markers of differentiated intestinal epithelium and myofibroblasts. RT-PCR demonstrated expression of α-smooth muscle actin and vimentin in myofibroblasts and E-Cadherin, CDX2, villin 1, intestinal alkaline phosphatase, chromogranin A, lysozyme, and Lgr5 in epithelial cells. These markers were maintained through several passages. CONCLUSION: Type I collagen gel supports long-term in vitro maintenance and expansion of fully elaborated human intestinal epithelium. Collagen-based methods yield familiar enteroid structures as well as a new pattern of sheet-like growth, and they eliminate the need for Matrigel for in vitro human intestinal epithelial growth. Future research is required to further develop this cell culture system for tissue engineering applications.

Use of collagen gel as an alternative extracellular matrix for the in vitro and in vivo growth of murine small intestinal epithelium.

Methods for the in vitro culture of primary small intestinal epithelium have improved greatly in recent years. A critical barrier for the translation of this methodology to the patient's bedside is the ability to grow intestinal stem cells using a well-defined extracellular matrix. Current methods rely on the use of Matrigel(™), a proprietary basement membrane-enriched extracellular matrix gel produced in mice that is not approved for clinical use. We demonstrate for the first time the capacity to support the long-term in vitro growth of murine intestinal epithelium in monoculture, using type I collagen. We further demonstrate successful in vivo engraftment of enteroids co-cultured with intestinal subepithelial myofibroblasts in collagen gel. Small intestinal crypts were isolated from 6 to 10 week old transgenic enhanced green fluorescent protein (eGFP+) mice and suspended within either Matrigel or collagen gel; cultures were supported using previously reported media and growth factors. After 1 week, cultures were either lysed for DNA or RNA extraction or were implanted subcutaneously in syngeneic host mice. Quantitative real-time polymerase chain reaction (qPCR) was performed to determine expansion of the transgenic eGFP-DNA and to determine the mRNA gene expression profile. Immunohistochemistry was performed on in vitro cultures and recovered in vivo explants. Small intestinal crypts reliably expanded to form enteroids in either Matrigel or collagen in both mono- and co-cultures as confirmed by microscopy and eGFP-DNA qPCR quantification. Collagen-based cultures yielded a distinct morphology with smooth enteroids and epithelial monolayer growth at the gel surface; both enteroid and monolayer cells demonstrated reactivity to Cdx2, E-cadherin, CD10, Periodic Acid-Schiff, and lysozyme. Collagen-based enteroids were successfully subcultured in vitro, whereas pure monolayer epithelial sheets did not survive passaging. Reverse transcriptase-polymerase chain reaction demonstrated evidence of Cdx2, villin 1, mucin 2, chromogranin A, lysozyme 1, and Lgr5 expression, suggesting a fully elaborated intestinal epithelium. Additionally, collagen-based enteroids co-cultured with myofibroblasts were successfully recovered after 5 weeks of in vivo implantation, with a preserved immunophenotype. These results indicate that collagen-based techniques have the capacity to eliminate the need for Matrigel in intestinal stem cell culture. This is a critical step towards producing neo-mucosa using good manufacturing practices for clinical applications in the future.