Methods for obtaining cells from lung tissue

Abstract

Methods for isolating lung cells are disclosed. Mechanical pressure can be used at one stage of the process to increase the yield of isolated cells, including alveolar type II cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 237,003, filed August 25, 2021, which is incorporated by reference in its entirety.

[0002] This application relates generally to cell isolation from tissue, and more specifically, but not exclusively, to methods and compositions for isolating lung cells from lung tissue, and to cells produced from such methods, which may be used for research, cell therapy, tissue engineering, and other applications. [Background technology]

[0003] Methods for isolating different cell populations from cadaveric tissues are usually specific to each cell population, often requiring different digestive enzymes, incubation times, and dissociation approaches. Due to the different isolation requirements for each cell type, when multiple cell types are required from the same organ, the tissue is often divided into separate pieces, thus reducing the overall potential yield for each cell type. Furthermore, these approaches are usually intensive and time-consuming, thereby further limiting the amount of tissue that can be processed while maintaining adequate cell health. As a result, it is a challenge to generate many different types of primary human cells from a single donor's tissue. This challenge is particularly relevant in the field of tissue engineering for both autologous and allogeneic applications, where cell number requirements are high and primary cells have limited proliferation capacity. These isolation limitations may also impact personalized medicine and drug development and screening, whereby in vitro models may require the generation of multicellular platforms from small pieces of donor tissue to achieve sufficient cellular complexity to accurately represent patient outcomes.

[0004] One specific example of the challenges of cell isolation stems from the processing of lung tissue. Alveolar type 2 (AT2) cells are notoriously difficult to isolate. AT2 cells are often isolated from the right middle lobe of the lung. Isolating AT2 cells using existing methods can take multiple researchers a full day and yield only a few hundred million cells. This lengthy, hands-on process often limits the isolation of other cell types that perform important functions in the lung. Thus, there is an unmet need for an isolation method that yields all the important cells of interest from donor lung tissue. Furthermore, the use of a single digestion method to isolate all cell types of interest increases the cell yield of each cell type. Summary of the Invention

[0005] Described herein is a method for isolating various cell types from donor organs using a single dissociation method.In some examples, disclosed is a method for isolating lung cells, such as one or more of alveolar type II cells (AT2), airway epithelial basal cells (AEP), interstitial cells, and endothelial cells, from human donor lung tissue.In some embodiments, the disclosed method allows for the isolation of a larger amount of cells than other existing methods.Disclosed herein is a method for tissue dissociation and a method for cell purification, which can allow for the isolation of a larger number of cells at one time.

[0006] Obtaining sufficient numbers of specific types of lung cells, such as AT2 cells, from human donor lung tissue has been a long-standing challenge. These cells can be used to support diagnostic testing, drug discovery and development, cell therapy, or the construction of engineered organs. AT2 cells can be isolated using a method originally developed by Leland Dobbs and optimized in the Sannes Laboratory at North Carolina State University ("Sannes method"; see Zhang, H., Newman, D. and Sannes, P. "HSULF-1 inhibits ERK and AKT signaling and decreases cell viability in vitro in human lung epithelial cells." Respiratory Research. 2012; 13(1): 69, which is incorporated herein by reference in its entirety). AT2 isolation using the Sannes method typically yields hundreds of millions of AT2 cells from one to two lobes of human donor lung tissue. In contrast, the methods disclosed herein may allow processing of all five lobes, which can produce as many as one billion AT2 cells without increasing staff or processing time.

[0007] Another major challenge is to isolate a large number of specific types of lung cells, such as AT2 cells, with sufficient purity for downstream expansion using Sannes or other published methods. For example, the purification approach of the Sannes method uses panning to remove leukocytes (differential adhesion of cells to a plate), followed by negative selection for fibroblasts. Panning approaches do not scale easily and therefore can be difficult with the increasing number of cells generated by the methods disclosed herein. Another existing purification method is the use of magnetic activated cell sorting (MACS) or fluorescence activated cell sorting (FACS)-based positive selection approaches based on AT2 cell surface marker HT2-280. However, this method is also not ideal because many fragile AT2 cells do not remain (average purification efficiency of 19% with MACS-based HT2-280 selection).

[0008] FIG. 1 shows an overview of a method for isolating specific cell types in an embodiment that relates to isolating AT2 cells, airway epithelial basal cells, and interstitial cells from a donor lung organ. It should be noted that this is just an embodiment, and similar methods should be possible to use to isolate other cell types from other types of organs. An embodiment of the present disclosure relates to isolating cells from a living tissue. These cells can be isolated by applying mechanical pressure to the tissue. In some embodiments, the living tissue can be lung tissue. In some embodiments, mechanical pressure can be applied after enzymatic digestion of the lung tissue. The living tissue can be crushed, for example, the living tissue can be crushed by the hand of a human operator until the distal tissue is liquefied. This method allows two or more lung lobes to be processed together. This can increase the amount of material that can be processed together. The isolation method can further include a filtration step. The total crude yield after digestion and filtration can be more than 30 billion cells (herein described as the post-filtration sample). The isolation method can include a purification step. The final purification yield of the method can be 1 billion or more AT2 cells. For airway epithelial basal cells, stromal cells, and endothelial cells, the method can include purification by culture selection. Culture selection can remove leukocytes.

[0009] Disclosed herein is a method of purifying cells from lung tissue. The method may include removing white blood cells. The method may include removing one or more other cell types. In some embodiments, antibodies bound to magnetic particles are used to select and remove white blood cells using magnetic activated cell sorting techniques. Antibodies bound to magnetic particles may be used to select and remove one or more other cell types. The remaining cells may be alveolar type II cells (AT2). The selected cells may be one or more of airway epithelial basal cells (AEP), interstitial cells, endothelial cells, among others. The method may include purifying a cell population of interest from a lung tissue isolate. The method may include removing white blood cells, interstitial cells, and airway epithelial basal cells. The method may include selecting endothelial cells. The method may include removing white blood cells and AT2 cells.

[0010] In some embodiments, cell surface proteins can be used to separate cells. The method can include using an antibody against at least one marker selected from CD45, CD16, CD32, CD90, CD144, CD31, CD140b, and CD271 to select less sensitive cells. The method can include using at least one marker selected from CD45, CD90, and CD271 markers to remove less sensitive cells. CD45, CD90, and CD271 beads can be used to remove white blood cells, stromal cells, and airway epithelial basal cells. In some embodiments, antibodies bound to magnetic particles are used to select and remove white blood cells, stromal cells, and airway epithelial basal cells. In some embodiments, a two-step selection can be performed, whereby CD45 selection is followed by a combination of CD90 and CD271 selection.

[0011] Disclosed herein is a method for forming engineered organs.The organs can be made from synthetic or natural lung matrices.The method can include seeding a scaffold matrix with cells obtained from the method disclosed herein.In one embodiment, engineered lung structures can be formed by seeding a lung scaffold with cells obtained from the method disclosed herein.

[0012] Disclosed herein is an engineered organ formed by seeding a scaffold with cells obtained from the method disclosed herein. Disclosed herein is an engineered lung structure formed by seeding a lung scaffold with cells obtained from the method disclosed herein. The cells can be purified by selecting leukocytes and at least one other cell type using antibodies against one or more cluster of differentiation (CD) markers. The CD markers can be one or more of CD45, CD16, CD32, CD31, CD90, CD144, CD140b, and CD271. In some embodiments, leukocytes, interstitial cells such as fibroblasts, endothelial cells, and airway basal cells can be selected. In some embodiments, the seeded cells can be one or more of alveolar type II cells, airway epithelial basal cells, pulmonary interstitial cells, and pulmonary endothelial cells.

[0013] As used herein, "Lung Crush Method" or "LCM" refers to a method that involves the application of mechanical force to disrupt tissue from which cells are to be isolated. The application of force can occur during or after enzymatic digestion of the tissue. The method may include additional steps, and the disruption force can be applied by any suitable means, such as mechanical grinding or by hand by a technician. [Brief description of the drawings]

[0014] [Figure 1]FIG. 1 shows a schematic of the cell isolation process from vital organs using the existing Sannes lab method (top panel), which consists of using scissors to dissociate tissue and positive selection to purify alveolar type 2 cells, compared to the method described herein (bottom panel, highlighted in yellow), which involves crushing and dissociating the tissue and negative selection to purify alveolar type 2 cells. [Diagram 2] FIG. 1 shows post-filtration (PF) yield per gram of tissue for Sannes versus LCM by donor compatibility comparison of the two tissue dissociation methods (n=3). [Diagram 3] FIG. 13 shows post-filtration (PF) AT2 purity (HT2-280+%) for Sannes versus LCM with donor compatibility comparison of the two dissociation methods (n=3). [Figure 4] Figure 1 shows the theoretical AT2 / g (tissue) for Sannes vs. LCM by donor compatibility comparison of the two dissociation methods (n=3). Theoretical AT2 / g is calculated as total cells / g multiplied by HT2-2 80%. [Diagram 5] FIG. 13 shows AT2 purity (HT2-280%) after selection of CD45 / CD90 / CD271 depleted AT2 samples for Sannes versus LCM with donor compatibility comparison of the two dissociation methods (n=3). [Figure 6] FIG. 1 shows AT2 after selection per gram of tissue for Sannes vs. LCM by donor compatibility comparison for the two dissociation methods (n=3). [Figure 7] FIG. 1 shows the average AT2 yield for each isolation method by donor compatibility comparison of the two dissociation methods (n=3). [Figure 8] FIG. 1 shows a comparison of the selection efficiencies of Sannes and LCM, calculated based on the actual AT2 yield after purification divided by the theoretical AT2 yield before purification. [Figure 9] FIG. 13 shows a summary of improvements in AT2 cell isolation when the LCM process was scaled to utilize all of the lung tissue from a single donor. [Figure 10]FIG. 1 shows a process schematic, highlighting one method of digesting and dissociating whole lung tissue, as well as purification methods that can be used to isolate airway epithelial basal cells, interstitial cells, AT2 cells, and endothelial cells from lung tissue. [Figure 11] FIG. 1 shows all four cell types isolated from one donor according to one embodiment. [Figure 12] FIG. 1 shows images from a lung crush technique. [Figure 13] FIG. 1 shows a summary of cell yield and purity from lungs when four different cell types (airway epithelial basal cells, interstitial cells, AT2 cells, and endothelial cells) were isolated from a single donor using the lung crush method. [Figure 14] FIG. 1 shows an example of airway epithelial basal cells obtained by the lung crush method. [Figure 15] FIG. 1 shows an example of interstitial cells obtained from the lung crush method. [Figure 16] FIG. 1 shows an example of endothelial cells obtained from the lung crush method. [Figure 17] FIG. 11 shows a schematic diagram of one embodiment of a process for obtaining airway epithelial basal cells, interstitial cells, AT2 cells, and endothelial cells from lung tissue using a different purification method compared to that described in FIG. [Figure 18] FIG. 1 shows a summary of AT2 isolation and characterization by the lung crush method and negative selection strategy described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Cells isolated from human or animal organs can be used to support in vitro diagnostic and pharmaceutical testing, cell therapy development, and cellularization of scaffolds for regenerative medicine. These cellularized scaffolds can be used for implantation into patients as clinical products. However, obtaining sufficient numbers of specific lung cell types has been a long-standing challenge in the field. One such example is the isolation of AT2 cells from human donor lung tissue for use in the formation of engineered lung tissue. The isolated AT2 cells can be banked, expanded, and used to cellularize porcine or 3D-printed lung scaffolds. These porcine or 3D-printed lung scaffolds can be implanted into patients. The isolated AT2 cells can also be used to support studies of AT2 cell identity and function, growth characteristics, pathology, and drug candidate screening in various platforms.

[0016] Isolation of AT2 cells has been performed using a method developed in the laboratory of Philip Sannes at North Carolina State University (herein referred to as the Sannes method). AT2 isolation using the Sannes method typically yields hundreds of millions of AT2 cells from one to two lobes of human donor lung tissue. Thus, the Sannes method must be repeated multiple times on different donors to ensure a billion or more cells, entailing high costs in time and materials. Furthermore, pooling of cells from different donors for human cells, tissues, and cell- and tissue-based products is restricted by the FDA.

[0017] In contrast, the method disclosed herein may allow processing of all five lung lobes, and may produce as many as one billion AT2 cells without increasing staff or processing time.Furthermore, the method disclosed herein may allow processing of larger quantities of cells, which may allow large-scale growth in bioreactors.The method disclosed herein may allow the production of increased numbers of cells, which may reduce the need to isolate cells from additional donors.The method disclosed herein may also allow the construction of donor-matched banks of multiple lung cell types, and may allow the repopulation of scaffolds with cells from a single donor.This is a key consideration for allogeneic tissue products, and the use of cells from a single donor with close HLA matching may be important to prevent organ rejection.

[0018] Furthermore, it has been difficult to isolate large numbers of AT2 cells or other specific lung cell types with sufficient purity for downstream propagation using the Sannes method or other published methods. A purification strategy that solves this problem is disclosed herein. This purification method uses negative selection to remove non-AT2 cells that may grow abnormally in downstream cultures, leaving sensitive AT2 cells in the negative population unlabeled for downstream use. The positively selected non-AT2 cells can be seeded in culture to generate a bank of other cell types of interest. Figure 1 shows an overview of a method for isolating specific cell types in an embodiment related to maximizing the number of AT2 cells isolated from donor lung organs with sufficient purity for downstream culture and propagation. It should be noted that this is just an embodiment and similar methods should be possible to use to isolate other cell types from other types of organs.

[0019] Figure 1 shows a schematic diagram of one embodiment of a method for cell selection from a living organ. In this embodiment, the living organ is a lung and the desired purified cells are AT2 cells. The lung is secured from a donor. The lung is purified, for example, by lavaging the airways with a buffer solution. The lung is then digested with an enzyme such as elastase or collagenase. At this point, the cellular tissue can be dissociated by either the Sannes method or the lung crush method.

[0020] The Sannes method (SM) may involve removing large white airways and large chunks of undigested tissue. Small pieces of digested tissue may be transferred to a cup, minced, and collected using three pairs of surgical scissors taped together (referred to as triple scissors). This process may be repeated several times until all of the digested tissue has been minced.

[0021] Lung crushing method (LCM) may involve applying a tissue crushing force using an object such as a hand or a mechanically automated crushing device, such as one using rollers in series or parallel, to crush the entire digested tissue all at once. Crushing may involve tearing open the pleura and allowing the digested tissue and cells to pour out and be collected in a container. Crushing may involve squeezing the digested tissue. Crushing may involve pulling the tissue apart. Crushing may involve squeezing the tissue and collecting additional cell suspension. The lung tissue may be crushed until only the airways and pleura remain. The airway tissue may be removed and the crushed tissue may be collected. There are few undigested pieces of tissue remaining in the cell suspension after crushing. In contrast, after cutting the tissue using the Sannes method, pieces of tissue ranging from approximately 1 to 5 mm are visible throughout the cell suspension. For example, tissue processed using LCM may have no more than 20%, 10%, 5%, 2%, or 1% by weight of tissue fragments that are 1 mm, 2 mm, or 5 mm or longer in diameter. In some embodiments, tissue processed using LCM contains no more than 5% by weight of tissue fragments that are 5 mm or longer in diameter. In some embodiments, tissue processed using LCM contains no more than 5% by weight of tissue fragments that are 2 mm or longer in diameter. In some embodiments, tissue processed using LCM contains no more than 5% by weight of tissue fragments that are 1 mm or longer in diameter. Determining the relative amount of tissue fragments of a particular size can be accomplished using appropriately sized sieves, meshes, and the like.

[0022] After dissociation, the collected liquid may be filtered. The liquid may be filtered through surgical gauze or mesh, silk, or nylon filters. The liquid may be filtered multiple times and through multiple filters. The liquid may also be centrifuged one or more times to resuspend the cell pellet.

[0023] Prior to scaling up LCM, three head-to-head isolations were performed on donor-compatible tissues to compare LCM to the Sannes method. For each donor in this comparison study, tissue was divided into left and right lungs. One lung from each donor was processed using the Sannes method and the other was processed using LCM. The lungs assigned to each process were varied for each donor, as well as the operator performing the isolation. Data from this comparison study are included in Figures 2-4.

[0024] Figure 2 shows the post-filtration (PF) total cell yield per gram of tissue for Sannes vs. LCM (n=3). These data demonstrate that the yield per gram of tissue is similar for the Sannes and lung crush methods. No statistically significant difference is observed in the amount of purified cells per gram of tissue (Welch's t-test, p<0.05).

[0025] Figure 3 shows post-filtration (PF) AT2 cell purity for Sannes vs. LCM after dissociation (n=3). No significant difference was found in post-filtration cell purity between the two dissociation methods (Welch's t-test, p<0.05).

[0026] Figure 4 shows the theoretical number of AT2 cells per gram of tissue for Sannes versus LCM, calculated as post-filtration yield multiplied by post-filtration purity (n=3). No statistically significant difference was evident in the theoretical yield of AT2 cells isolated from tissue using the two different dissociation methods (Welch's t-test, p<0.05).

[0027] After filtration, the desired cells may be purified. The selection process may be a negative or positive selection process. In the Sannes purification method, undesirable cells may be removed by differential adhesion to non-tissue culture petri dishes with or without the use of antibodies. In some embodiments, a combination of differential adhesion and magnetic removal may be used. The Sannes AT2 purification process may involve plating and panning to remove stromal cells such as white blood cells and fibroblasts. The purification process may involve using antibodies such as AS02 antibodies to selectively bind to stromal cells. The antibodies may be bound to metal particles, allowing for magnetic removal of stromal cells. Another commonly used method is to positively select cells using antibodies against HT2-280, an AT2 cell surface marker (Terrace Biotech, a mouse IgM monoclonal antibody), followed by staining with anti-mouse IgM magnetic beads. While positive selection via HT2-280 produces highly pure samples with low levels of contaminating cell types that can cause the culture to grow abnormally, purification efficiency is low using this selection method, thus resulting in low overall AT2 yields.

[0028] In the purification method described herein, other non-AT2 cells can also be removed.For example, CD45, CD90 and CD271 antibodies can be added to bind to white blood cells, interstitial cells and airway basal cells.These antibodies can be bound to metal particles.The metal particles, antibodies and bound blood cells, interstitial cells and airway basal cells can be magnetically removed.

[0029] Antibodies bound to magnetic particles can also be used to select and remove one or more cell types, leaving the most sensitive desired cells. The remaining desired cells in the negative population can be stored in a bank for later use. The isolated cells can be alveolar type II cells (AT2). The isolated cells can be one or more of airway epithelial basal cells, interstitial cells, endothelial cells, among others.

[0030] Depending on the identity of the desired cells, the method may include removing leukocytes, interstitial cells, and / or airway epithelial basal cells. The method may include removing leukocytes and / or alveolar type II cells. Magnetic beads coupled to antibodies against cell surface proteins may be used to selectively separate cells that are not AT2 cells. The selected cells may be removed using at least one antibody against a cell surface protein selected from CD45, CD16, CD32, CD90, CD31, CD144, CD140b, and CD271. The selected cells may be removed using at least one antibody against a cell surface protein selected from CD45, CD90, and CD271 markers. CD45, CD90, and CD271 antibodies may be used for leukocytes, interstitial cells, and airway basal cells from the AT2 population. Alternative markers may be used to remove all cells from the sample except the desired cells.

[0031] A negative selection approach using magnetic beads coupled to antibodies against CD45, CD90, and CD271 was implemented to purify AT2 cells by direct competitive isolation assays of LCM and the Sannes dissociation method presented in Figures 2-4. Post-purification data from these comparisons are included in Figures 5-8.

[0032] Figure 5 shows AT2 purity (% positive cells for HT2-280) after purification of CD45 / CD90 / CD271 depleted samples by Sannes vs. LCM comparison. No statistically significant difference was observed between the two samples (n=3 donors, Welch's t-test, p<0.05).

[0033] Figure 6 shows the number of purified AT2 cells per gram of tissue by Sannes vs. LCM comparison. No statistically significant difference was observed between the two samples (n=3 donors, Welch's t-test, p<0.05).

[0034] Figure 7 shows the average total AT2 cell yield after purification for each Sannes and LCM isolation method performed on similar amounts of tissue (Donor 1: Sannes 400g, LCM 380g; Donor 2: Sannes 244g, LCM 220g; Donor 3: Sannes 306g, LCM 301g). As expected given that the same amount of tissue was processed and the same purification strategy was performed, no statistically significant differences were found between the two samples (n=3 donors, Welch's t-test, p<0.05).

[0035] Figure 8 shows a comparison of the selection efficiency between Sannes and LCM, calculated based on the post-purification AT2 yield divided by the theoretical pre-purification AT2 yield. The lung crush method provided a statistically significant improvement in selection efficiency when compared to the Sannes method (n=3 donors, Welch's t-test, p<0.05).

[0036] After a head-to-head competitive comparison, LCM was scaled up to process both left and right lung tissue (bilateral lungs) from donors. The triple-scissor dissociation step for the Sannes protocol is laborious and time-consuming, thereby limiting the amount of tissue that can be processed at one time. A simplified dissociation method using the lung crush technique allows for the handling of entire lung tissue from a single donor at once, decreasing overall processing time.

[0037] FIG. 9 shows a summary of the improvements seen in one embodiment of the method disclosed herein. In this embodiment, AT2 cells were isolated from donor lung tissue using a scaled-up lung crush method compared to previous Sannes (including triple scissor minced samples) data. AT2 cells were then purified using negative magnetic bead selection and compared to previous data using other established purification methods. The lung crush dissociation approach significantly increased (a) tissue processing capacity and (b) the number of unpurified AT2 cells in the post-digestion sample (theoretical AT2 cell yield). This data set encompassed all full-scale LCM runs in which both lungs (all lobes) were processed. (c) Various purification strategies were compared for cells isolated by Sannes triple scissor. CD45 depletion tended to improve purification efficiency compared to HT2-280 selection. However, HT2-280 selection produced cells with sufficient purity for downstream AT2 culture, whereas CD45 depletion alone did not. Therefore, a CD45 / CD90 / CD271 depletion method was established to deplete basal cells (CD271+) and fibroblasts (CD90+) along with CD45+ cells using negative selection by surface markers. (d) Comparison of final AT2 yields generated from the various isolation and purification approaches. The average AT2 yield per donor was 73M using the original method (Sannes, HT2-280 selection performed on a small-scale MACS instrument). The Sannes method combined with CD45 depletion or CD45 / CD90 / CD271 depletion tended to increase the AT2 yield. However, the lung crush method combined with CD45 / CD90 / CD271 produced the highest AT2 yield. Furthermore, considering the higher cell yield, a large-scale CliniMACS purification instrument was required for full-scale processing. This data set includes a full-scale run of LCM, where both lungs were processed and purified on the CliniMACS using exhaustion tubing, where no process errors or deviations occurred.Combining these new methods demonstrated a statistically significant increase to an average of 930 million AT2 cells isolated from a single donor (t-test, ANOVA for a and b, Tukey's multiple comparison test for c and d). * p<0.05, *** p<0.001, **** p<0.0001).

[0038] These purification methods are summarized in the table below:

[0039] [Table 1]

[0040] [Table 2] EXAMPLES

[0041] The following examples describe certain aspects of some embodiments of the present disclosure in order to illustrate and provide a description for those skilled in the art. The examples should not be construed as limiting the disclosure, as they merely provide certain methodologies useful in understanding and practicing some embodiments of the disclosure.

[0042] [Comparative Example 1] Dissociation and purification using the Sannes method In this method, a lobe (usually the right middle lobe) is dissected for processing.

[0043] Clearance: The lobar vasculature was perfused free of blood with solution II (aqueous solution of NaCl, Na2HPO4, HEPES, CaCl2, and MgSO4 7H2O) at 37° C. Air was removed from the lobe, the lobe was cannulated, and irrigated with solution I (aqueous solution of NaCl, Na2HPO4, HEPES, glucose, and EGTA). The irrigation was repeated until the draining solution ran clear.

[0044] Digestion: Elastase was dissolved in solution II at 37° C. Lung lobes were incubated in a water bath set at 37° C. and filled with warm elastase solution. Lungs were digested until they were sufficiently loose.

[0045] Tissue dissociation (Sannes method): Large undigested chunks of tissue were excised. Large white airways were removed and discarded. Smaller leaf pieces were added to a cooling cup on ice containing 5 mL of cold DNase solution (DNase 25 mg in 50 mL of solution II). These smaller leaf pieces were batch minced using three pairs of surgical scissors held together in tandem or taped together, "triple scissors". The minced cell solution was collected in a 1 liter flask chilled on ice. Once all the tissue was processed, FBS was added to the cell suspension and the flask was vigorously shaken in a water bath (37°C) for 3 minutes.

[0046] Filtration: The cell suspension was filtered through a layer of moistened surgical gauze up to three times. The cell suspension was filtered through two layers of moistened surgical gauze. This was repeated at least once to remove most of the large tissue debris. The cell suspension was then filtered once or twice through a triple layer of moistened gauze. The cell suspension was filtered through a 165 μm silk or nylon mesh.

[0047] Centrifugation: The cell suspension was centrifuged at 200×g for 10 minutes at 4° C. The supernatant was discarded and the cell pellet was resuspended in 5 mL of DMEM medium.

[0048] Plating: Petri dishes were prepared with 500 μg / mL human IgG in Tris buffer, pH 9.5. In some cases, the dishes were incubated overnight at 4° C. Approximately 5 mL of cell solution was delivered to the prepared IgG dishes.

[0049] Panning: The prepared cell dish was panned in the incubator for up to 1 hour until the leukocytes were well attached and appeared gray, but the AT2 cells were still refractile and did not bind. The fibroblasts also started to bind. The cell dish was removed from the incubator and gently rocked to dislodge the AT2 cells. The unbound cell solution was collected and centrifuged at 200×g for 10 minutes at 4° C. The supernatant was discarded.

[0050] Fibroblast Depletion Option 1-Differential Adhesion: Fibroblast populations were depleted by differential adhesion to non-tissue culture treated Petri dishes for approximately 1 hour.

[0051] Fibroblast depletion option 2 - magnetic depletion: Fibroblast populations were depleted using an AS02 anti-fibroblast antibody negative selection step. The cell pellet was resuspended in DMEM. The AS02 antibody was used to selectively adhere to fibroblasts. The tube of cells and antibody was gently rolled for a 10 minute incubation time at 4°C. DMEM / 0.1% cell culture grade BSA was added and the solution was centrifuged (10 minutes, 800 rpm, 4°C). The supernatant was removed and the cells were resuspended in DMEM / 0.1% BSA.

[0052] Dynabeads preparation: Pan-mouse IgG Dynabeads were washed in 1 mL of DMEM / 0.1% BSA, magnetically collected, and resuspended in DMEM / 0.1% BSA. Dynabeads were added to the cells, and the solution was incubated at 4° C. for 30 minutes and gently rolled end to end. Fibroblasts were magnetically removed by a DynaMag-15 magnet for approximately 2 minutes. Unbound AT2 cells were poured off, collected, and counted. Cells were concentrated by centrifugation, and the medium was replaced for seeding. The pellet was resuspended in DMEM with 10% FBS and 2× antibiotic / antimycotic. Cells were counted and stored for later use.

[0053] Fibroblast depletion option 3: Fibroblasts were depleted using a combined method of option 1 and option 2.

[0054] [Example 2] Isolation of AT2 cells, airway epithelial basal cells, and interstitial cells using the lung crush method and CD45 / CD90 / CD271 depletion In this method, both lungs (all lobes) are used for processing.

[0055] Cleansing: The lung airways were cannulated and infused with HBSS (-MgCl2, -CaCl2). HBSS was expelled from the lungs by gentle massage. The lavage was repeated three times. Two final rinses were completed with HBSS (+MgCl2, +CaCl2).

[0056] Digestion: Elastase, collagenase type IV, calcium chloride, and DNase were dissolved in HBSS (-MgCl2, -CaCl2) at 37°C and infused into the lungs (in Comparative Example 1, collagenase type IV and DNase were not used). The lungs were placed in a Whirlpak bag and placed in a water bath set at 37°C and the lungs were allowed to digest for approximately 45 minutes.

[0057] Tissue dissociation (lung crush technique): wearing sterile gloves, a human operator placed their hand into the bag. The pleura was torn open and the lung tissue was pulled apart and manually crushed until only the airways remained. At this point, the remaining airway tissue was removed from the bag and discarded. The liquid contents of the Whirlpak bag were collected.

[0058] Filtration: The cell suspension was filtered through a series of mesh sheets with decreasing pore size (2000 μm, 1000 μm, 200 μm, 100 μm). After filtration, the cell suspension was grown in DMEM / F12 medium with DNase. 5% FBS was added to the cell suspension and mixed.

[0059] Centrifugation: The cell suspension was centrifuged at 300×g for 8 min. The supernatant was discarded and the cell pellet was resuspended in 5 mL of DMEM / F12 with DNase.

[0060] Once the cell suspension was obtained according to the above method, AT2 cells, AEP cells, and stromal cells were purified according to the following method.

[0061] Magnetic bead labeling of interstitial cells, airway basal cells, and leukocytes: Cells were counted, for example, using a K2 Cellometer (Nexcelom). The cell suspension was centrifuged, for example, at 300×g for 5 minutes at 4° C. The cell suspension was resuspended in culture medium. In some cases, the culture medium contained DNase. CD45, CD90, and CD271 beads were added to bind leukocytes, fibroblasts, and airway basal cells, respectively. In some embodiments, beads were added in excess relative to the expected number of interstitial cells, airway basal cells, and / or leukocytes in the sample. The sample was mixed thoroughly and incubated. In some embodiments, incubation was performed at room temperature for 45 minutes. The cells were washed, centrifuged, and resuspended in culture medium.

[0062] Magnetic separation of AT2 cells from stromal cells, airway epithelial basal cells, and white blood cells: Cells are placed in a container such as a blood transfer bag and attached to a CliniMACS tubing set. Depletion program, specifically the depletion program 3.1 program in the CliniMACS™ system (cell purification system) in this example, is selected. Cells that are not selected using the depletion program (AT2 cells) are counted via a K2 Cellometer™ (cell counter) and stored for later use.

[0063] Split the selected cells (CD45+ / CD90+ / CD271+) and culture at approximately 300,000-400,000 cells / cm in separate flasks in culture medium designed to support airway epithelial basal or stromal cells. 2 These cultures generated purified populations of AEP and stromal cells over passages.

[0064] [Example 3] Interstitial purification by culture selection after tissue dissociation using the lung crush method Lung cells were isolated from lung tissue according to the following method. Donor lung tissue was cleaned and digested according to the method disclosed herein or known to those skilled in the art. Lung tissue was dissociated using a method such as the lung crush method. Cell suspension was filtered through surgical gauze, nylon, mesh, or other porous material according to the method disclosed herein or other methods known in the art.

[0065] Once the cell suspension was obtained as described above, stromal cells were purified as described below.

[0066] The filtered samples were frozen. Samples taken after isolation were assessed for CD90 expression. The filtered cells were thawed and 3,000 CD90+ cells / cm were counted. 2 The cultures were seeded in stromal cell medium at a concentration of 1000 mM NaCl, 100 mM MgCl, 1 ... and 100 mM MgCl. These cultures generated purified populations of stromal cells over passages. This purification method serves as an alternative to the use of selected cells to generate stromal cell cultures, allowing for maximization of the selected population to be used to generate airway epithelial basal cell cultures.

[0067] [Example 4] Isolation of endothelial cells by positive selection after lung crush method Endothelial cells were isolated from lung tissue according to the following method. Donor lung tissue was cleaned and digested according to the method disclosed herein or known to those skilled in the art. Lung tissue was dissociated using a method such as the lung crush method. Cell suspension was filtered through surgical gauze, nylon, mesh, or other porous material according to the method disclosed herein or other methods known in the art.

[0068] Once the cell suspension was obtained according to the above method, endothelial cells were selected according to the following method.

[0069] Magnetic bead labeling and endothelial cell isolation: Cells were counted, for example, using a K2 Cellometer (Nexcelom). The cell suspension was centrifuged, for example, at 300×g for 5 minutes. The cell suspension was resuspended in culture medium. In some cases, the culture medium contained DNase. CD45 beads were added to the cell suspension to bind to the white blood cells. In some embodiments, the incubation was for 15 minutes. The cells were placed in a container, such as a blood transfer bag, and attached to a CliniMACS tubing set. A depletion program was utilized to select for CD45 positive white blood cells. CD31 beads were then added to the negative fraction from the first purification step to bind to the endothelial cells. In some embodiments, the beads were added in excess relative to the expected number of endothelial cells in the sample. The sample was mixed thoroughly and incubated. In some embodiments, the incubation was for 15 minutes at room temperature. The cells were washed, centrifuged, and resuspended in culture medium. The endothelial cells were then selected using a MultiMACS instrument. The selected endothelial cells were counted via K2 Cellometer and seeded into cultures in endothelial cell culture medium.

[0070] Endothelial cells can also be obtained by seeding cells directly into culture after digestion followed by purification using CD31 selection after 1-2 passages in culture.

[0071] [Example 5] Isolation of four cell types from one donor FIG. 10 shows a schematic diagram of one embodiment of the process, highlighting the method used to digest and dissociate all of the lung tissue, as well as the purification method used to separate airway epithelial basal cells, interstitial cells, AT2 cells, and endothelial cells from the lung tissue. All of the lung lobes were digested and dissociated using the lung crush method. To generate a purified population of interstitial cells, the post-filtered sample was seeded directly into interstitial medium and grown for three passages (P2). The post-filtered cells at 20B were stained with CD45 beads and purified using the Depletion 3.1 program on a CliniMACS. The depleted sample from this first purification was then stained with CD90 and CD271 and purified on a MultiMACS instrument. The depleted cell sample from the second purification step was referred to as the AT2 population. The selected cell sample from the second purification step was seeded into culture in airway epithelial basal cell growth medium and further purified via culture for three passages (P2). To generate endothelial cells, separate aliquots of the filtered samples were first stained with CD45 beads, depleted on the CliniMACS, and subsequently stained with CD31 beads and purified via positive selection on the MultiMACS instrument. Selected samples were seeded into cultures in endothelial cell medium and grown for 4 passages (P3).

[0072] FIG. 11 shows the results of isolation of four lung cell types (AT2, endothelial, interstitial, and airway epithelial basal) from one donor described in FIG. 10. FIG. 11(a) shows tissue isolation (weight and total cell yield after filtration) and total AT2 yield information, and FIG. 11(b) shows morphological images of each of the four cell types. AT2: 24 hours after seeding, 20x objective; endothelial: passage 3, 10x objective; interstitial and airway epithelial: passage 2, 10x objective. FIG. 11(c) shows the purity of each of the four cell populations as shown by flow cytometry (AT2, HT2-280 expression after CD45 / CD90 / CD271 depletion; endothelial, CD144 expression at passage 3; interstitial, CD90 expression at passage 2; AEP, CK5 expression at passage 2). FIG. 11(d) presents the growth characteristics of endothelial (passage 3), stromal (passage 2), and AEP (passage 2) cultures.

[0073] FIG. 12 is a set of photographs taken of the lung crush procedure being performed on digested lung tissue to demonstrate the process. FIG. 12(a) shows a series of images showing the lung pleura being torn apart to release the digested tissue and cells into a collection bag. FIG. 12(b) shows a series of images showing the lung tissue being squeezed to release the cells into a collection bag. FIG. 12(c) shows images of two different lungs after the lung crush procedure, showing that minimal remaining lung tissue can be seen after the lung crush dissociation is complete. FIG. 12(d) shows an image of the resulting cell suspension collected by the lung crush procedure, showing minimal intact lung tissue fragments.

[0074] Figure 13 summarizes AT2 cell isolation and characterization. Figure 13(a) shows AT2 yield (total viable cell yield x percent HT2-280 positive cells) and Figure 13(b) shows purity (percent HT2-280 positive cells) in 15 isolations in which both lungs were digested and dissociated using the lung crush method, purified using magnetic beads, and depleted for CD45, CD271, and CD90 on the CliniMACS instrument using the depletion tubing set, including runs in which two-step purification on the CliniMACS and MultiMACS was performed as described in Figure 10, and excluding runs with process errors. The average AT2 yield was 930e6 cells, and the average AT2 purity was 70%. Figure 13(c) shows an example flow cytometry dot plot of isolated and purified AT2 cells from one donor, confirming expected marker expression using HT2-280 and pro-SP-C antibodies (black: target antibody, purple: isotype control). Figure 13(d) shows real-time PCR analysis of AT2 gene expression for two donors normalized to alveolar type 1-like (AT1-like) cell gene expression. AT1-like cells were generated by culturing AT2 cells for 7 days in a medium intended to promote AT1 conversion. RNA was isolated from samples using a QIAGEN RNeasy Mini Kit. cDNA was generated and real-time PCR was performed using probes for genes of interest. These data demonstrate the expression of several expected AT2 genes in AT2 cells isolated using the methods described herein, including SFTPB, SFTPC, SFTPD, LAMP3, ABCA3, and NAPSA.

[0075] Figure 14 shows a summary of airway epithelial basal cell isolation and proliferation from donors where airway epithelial basal cells were grown from selected fractions of CD45 / CD90 / CD271 depletion following lung crush dissociation as described in Example 2. CD45 / CD90 / CD271 selected cells were frozen on the day of isolation, then thawed and seeded in airway cell medium to initiate culture. Cells were grown for a total of three passages (passage 0 to passage 2). The table in Figure 14(a) shows metrics of airway epithelial basal cell proliferation. At every passage, culture area, number of cells / cm at harvest, 2 , total cell number harvested, fold change, population doubling number, population doubling level, and population doubling time were collected. More than 1 billion airway basal epithelial basal cells were generated from this donor after only one passage in culture (passage 0). While the size of the subsequent cultures at passages 1 and 2 was not maximized, the proliferation potential of the basal cells over further passages was demonstrated. The fold change of cells at passages 1 and 2 was 70.1 and 43.0, respectively. The population doubling times at passages 1 and 2 were 23.7 hours and 26.4 hours, respectively. Figure 14(b) shows the expression of airway epithelial basal cell markers cytokeratin 5 (ck5) and tumor protein 63 (p63) measured by flow cytometry from passage 0 to passage 2, demonstrating the maintenance of basal cell identity during the expansion. More than 80% of the population expressed both markers from passage 0 to passage 2. Basal cells from conducting airway are usually isolated through digestive airway tissue segments, followed by scraping airway lumen.The advantage of the method described herein is that it can simply collect cells from selected fractions of the purification approach used to isolate AT2 cells, without the need to carry out separate cell isolation process and without sacrificing any AT2 cells.

[0076] Figure 15 shows a summary of stromal cell proliferation from donors in which stromal cells were grown by seeding filtered cells collected by lung crush method in stromal cell medium as described in Example 3. Filtered cells were frozen on the day of isolation and then thawed to initiate stromal culture. Cells were grown for a total of three passages (passage 0 to passage 2). Figure 15(a) shows metrics of stromal cell proliferation. At every passage, culture area, number of cells / cm at harvest. 2 , total cell number harvested, fold change, population doubling number, population doubling level, and population doubling time were collected. The cell yield at passage 0 was 72 million cells, and expansion continued for two additional passages, with a decrease in doubling time at passages 1 and 2 (28.6 and 30.5 hours, respectively, compared to 57.6 hours at passage 0), indicating an increase in cell growth rate. Figure 15(b) shows the expression of stromal cell markers CD90 and CD140b measured by flow cytometry over the course of expansion, demonstrating that stromal cell identity was maintained. CD140b expression was low at passage 0, while at passages 1 and 2, more than 80% of the cells expressed both CD90 and CD140b. The average yield after filtration from processing both lungs using the lung crush dissociation method is approximately 35 billion cells. Thus, 20-30 B of post-filtration cells can be allocated for AT2 purification while leaving excess post-filtration sample behind to seed stromal cell cultures, without sacrificing many of the AT2 or airway epithelial basal cells.

[0077] Figure 16 shows a summary of the isolation and proliferation of donor-derived endothelial cells, in which endothelial cells were grown by seeding cells after lung crush filtration as described in Example 4. The cells after filtration were frozen on the day of isolation, then thawed and seeded in endothelial cell growth medium to initiate endothelial culture. Cells were successfully grown for a total of 5 passages (from passage 0 to passage 4), and CD31 magnetic bead selection was performed after harvest at passage 1. The table in Figure 16(a) shows a summary of endothelial cell proliferation. At every passage, culture area, cell number / cm at harvest. 2, total cell number harvested, fold change, population doubling number, population doubling level, and population doubling time were collected. Figure 16(b) shows expression of CD144, a marker of endothelial cells, measured by flow cytometry after every passage, including pre- and post-purification, starting from passage 1. After CD31 magnetic bead selection after passage 1, CD144 expression was maintained at greater than 80%.

[0078] [Example 6] Isolation of four cell types from a single donor using an alternative purification approach FIG. 17 shows a schematic diagram of a different embodiment of the process to obtain four different cell types (AT2 cells, airway epithelial basal cells, stromal cells, and endothelial cells) from one donor. Digestion and tissue dissociation are the same as those described in Example 5, but a different purification approach for AT2 and endothelial cells is described in this embodiment. To generate a purified population of AT2 cells, 20-30B of post-filtration cells are stained with CD45, CD90, and CD271 beads together in one step and run through a depletion column on a CliniMACS instrument without any subsequent purification on a MultiMACS instrument. The depleted cell sample is referred to as the AT2 population. To generate a purified population of airway epithelial basal cells, as described in Example 5, the selected fraction is seeded in basal cell growth medium. To generate a purified population of endothelial cells, a portion of the post-filtration sample is seeded directly in endothelial cell medium on the day of isolation. The cells are then harvested after one passage and purified using a MACS-based CD31 selection approach, as described in Example 4. The endothelial cells are then returned to culture for subsequent expansion or experimentation. Finally, a portion of the filtered sample is seeded in stromal cell growth medium, as described in Example 3, to generate stromal cells.

[0079] FIG. 18 shows a summary of isolated cells from lungs where the lung crush method was used to attempt isolation of four different cell types (AT2: alveolar type 2, AEP: airway epithelial basal, endothelial, and interstitial) from one donor. The table in FIG. 18(a) shows the total cell yield, cell purity, and passage number associated with the yield and purity reported for each cell type. For all cell types in the table, the total yield is the number of viable cells counted at the time of harvest from the reported passage, and the purity is the percent of the cell type of interest in the total yield measured via flow cytometry. HT2-280 expression was used to determine AT2 cell purity. Ck5 expression was used to determine airway epithelial basal cell purity. CD144 expression was used to determine endothelial cell purity. CD90 expression was used to determine interstitial cell purity. Various purification strategies according to lung crush method and cell culture media were used for the results presented in this table. An asterisk next to the total yield indicates that the isolation of that cell type was not maximized for that donor, and therefore it is predicted that the total cell yield may have been higher. The designation of "early" passage for AT2 cells indicates that the AT2 cells were not expanded prior to the analysis of total yield and purity. The cell culture after isolation was used to further purify the other three cell types (AEP, endothelial, and stromal), which is why they were included at higher passages. Finally, an X in the total yield column indicates that the isolation of that cell type was unsuccessful for that donor due to microbial contamination, lack of cell growth, or abnormal growth of a different cell type. Figure 18(b) shows a breakdown of the causes for each of the unsuccessful isolation attempts mentioned in Figure 18(a).

[0080] As used herein, the singular terms "a," "an," and "the" may include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to an object may include multiple objects unless the context clearly dictates otherwise.

[0081] As used herein, the terms "substantially" and "approximately" are used to describe and explain small variations. When used in conjunction with an event or situation, this term can refer to when the event or situation occurs exactly, as well as when the event or situation occurs approximately. When used in conjunction with a numerical value, this term can refer to a range of variation of less than or equal to ±10% of the numerical value, for example, less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%. When referring to a first numerical value as being "substantially" or "about" the same as a second numerical value, the term can refer to the first numerical value being within a range of variation of less than or equal to 10% of the second numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%.

[0082] Furthermore, amounts, ratios, and other numerical values ​​are sometimes presented in range format herein.It is understood that such range format is used for convenience and conciseness, and should be understood to be flexible to include not only the numerical values ​​explicitly specified as the limits of the range, but also to include all individual numerical values ​​or subranges contained within the range as if each numerical value and subrange were explicitly specified.For example, a ratio range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and subranges such as about 10 to about 50, about 20 to about 100, etc.

[0083] Although the present disclosure has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various modifications may be made and equivalents substituted without departing from the true spirit and scope of the disclosure as defined by the appended claims. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, method, operation(s) to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto. In particular, while a particular method may be described with reference to certain operations performed in a particular order, it will be understood that these operations may be combined, sub-divided or rearranged to form equivalent methods without departing from the teachings of the present disclosure. Thus, unless otherwise indicated herein, the order and grouping of operations are not limitations of the present disclosure.

Claims

1. 1. A method for isolating cells from lung tissue, the method comprising applying mechanical pressure to said lung tissue.

2. 10. The method of claim 1, wherein said application of mechanical pressure results in disrupted tissue having no more than 10% by weight of tissue fragments of 5 mm or larger in size.

3. 10. The method of claim 1, wherein said application of mechanical pressure results in disrupted tissue having 5% by weight or less of tissue fragments 5 mm or larger in size.

4. 10. The method of claim 1, wherein said application of mechanical pressure results in disrupted tissue having 1% by weight of tissue fragments of 5 mm or larger in size.

5. The method of claim 1 , wherein the lung tissue is crushed.

6. The method of claim 5 , wherein the crushing comprises crushing in the hands of a human operator.

7. 10. The method of claim 1, wherein more than two lung lobes are processed together.

8. The method of claim 1 further comprising enzymatic digestion.

9. 9. The method of claim 8, wherein the enzymes used in the enzymatic digestion include elastase.

10. 10. The method of claim 9, wherein the enzymes used in the enzymatic digestion further comprise type IV collagenase.

11. The method of claim 1 , further comprising filtering the disrupted lung tissue.

12. 10. The method of claim 1, further comprising purifying the disrupted lung tissue.

13. 13. The method of claim 12, wherein the purification step comprises removing white blood cells and at least one other cell type, and wherein the one other cell type is not a white blood cell.

14. The method of claim 1 , wherein the cells are alveolar type II cells.

15. The method of claim 1 , wherein the cells are airway epithelial basal cells.

16. The method of claim 1 , wherein the cells are stromal cells.

17. The method of claim 1 , wherein the cells are endothelial cells.

18. A cell isolated by the method of any one of claims 1 to 17.

19. 10. The method of claim 1, wherein the final purified yield of said method is at least 1 billion cells.

20. A method for purifying a cell population of interest from a lung tissue isolate, comprising removing leukocytes and at least one other cell type, wherein the one other cell type is not a leukocyte.

21. 20. The method of claim 19, wherein antibodies bound to magnetic particles are used to select and remove said white blood cells and said at least one other cell type.

22. The method of claim 1, comprising selecting leukocytes, stromal cells, and airway epithelial basal cells.

23. 18. The method of any one of claims 1 to 17, comprising selecting leukocytes, stromal cells, airway epithelial basal cells, and endothelial cells.

24. 22. The method of any one of claims 15, 16 or 21, wherein at least one antibody against a cell surface protein selected from CD45, CD16, CD32, CD90, CD31, CD144, CD140b, and CD271 cell surface proteins is used to select and remove non-AT2 cells.

25. 23. The method of any one of claims 15, 16, 19 or 22, wherein antibodies against CD45, CD90 and CD271 cell surface proteins are used to remove leukocytes, interstitial cells and airway basal cells.

26. 23. The method of any one of claims 15, 16, 19 or 22, wherein antibodies against CD45, CD90, CD31 and CD271 cell surface proteins are used to remove leukocytes, interstitial cells, endothelial cells and airway basal cells.

27. 22. The method of claim 21, wherein CD31 is used to select endothelial cells.

28. 16. The method of claim 15, further comprising purification by culture selection.

29. 29. The method of claim 28, wherein the culture selection removes leukocytes.

30. 1. A method for isolating cells, comprising: applying mechanical pressure to lung tissue; removing leukocytes and at least one other cell type from the disrupted lung tissue; wherein said one other cell type is not a leukocyte.

31. 31. The method of claim 30, wherein said application of mechanical pressure results in disrupted tissue having no more than 10% by weight of tissue fragments of 5 mm or larger in size.

32. 31. The method of claim 30, wherein said application of mechanical pressure results in disrupted tissue having no more than 5% by weight of tissue fragments 5 mm or larger in size.

33. 31. The method of claim 30, wherein said application of mechanical pressure results in disrupted tissue having 1% or less by weight of tissue fragments 5 mm or larger in size.

34. 10. A method of forming an engineered lung structure, comprising seeding a lung scaffold with cells obtained from the method of claim 1.

35. 33. The method of claim 32, wherein the cells from the method of claim 1 are separated from other cells after a step using one or more CD markers to remove leukocytes and at least one other cell type from the cell suspension.

36. 36. The method of claim 35, wherein at least one antibody against a CD marker from CD45, CD16, CD32, CD144, CD31, CD90, CD140b, and CD271 is used.

37. 32. The method of claim 30 or 31, wherein leukocytes, fibroblasts, and airway basal cells are removed.

38. 33. The method of any one of claims 30 to 32, wherein the seeded cells are alveolar type II cells.

39. 33. The method of any one of claims 30 to 32, wherein the seeded cells are airway basal cells.

40. 33. The method of any one of claims 30 to 32, wherein the seeded cells are lung interstitial cells.

41. 33. The method of any one of claims 30 to 32, wherein the seeded cells are pulmonary endothelial cells.

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