Method for culturing cartilage and its spheroids

Abstract

To provide spheroids useful for autologous chondrocyte implantation (ACI) products. [Solution] Chondrocytes are isolated from human or animal tissue, spread as a monolayer, cultured in 2D, harvested, cultured in a suspension in a 3D environment, and the resulting spheroids are selected. The total culture time for 2D and 3D is 55 days or less, calculated from the start of chondrocyte isolation, with the 2D culture time preferably exceeding 10 days.

Description

[Technical Field]

[0001] The present invention relates to a method for growing or increasing chondrocytes and providing spheroids thereof, which spheroids are useful in Autologous Chondrocyte Implantation (ACI) products. Accordingly, the present invention particularly relates to the production of spheroids from articular cartilage and their use. [Background technology]

[0002] Although articular cartilage exhibits remarkable resilience, this tissue is unable or almost unable to repair itself, and untreated lesions can lead to osteoarthritis (OA). This low natural regenerative capacity has led to the development of cell therapies such as autologous chondrocyte implantation (ACI), which aim to provide painless functional repair of articular cartilage defects. However, this type of method does not ensure cartilage regeneration, and no adequate long-term studies have been conducted to date. Therefore, there is a strong need for cartilage regeneration methods in young, active patients, for example, who have traumatic lesions or who also have cartilage degeneration pathologies.

[0003] Many studies have been performed using chondrocytes isolated from bovine, rabbit, or ovine cartilage. However, the data obtained and animal-based concepts of this type cannot be extrapolated to the human case. Detailed biochemical and molecular studies using human chondrocytes have been hindered by a series of factors, including the lack of availability of human tissue, which is associated with the very low number of cells available in biopsies, their limited proliferative potential, and the high phenotypic instability of cultured chondrocytes.

[0004] Chondrocytes are cells that originate from chondroblasts and are formed in cartilage tissue. Together with the intercellular matrix (extracellular matrix (ECM)), chondrocytes form the main component of cartilage.

[0005] Natural articular cartilage tissue has an extracellular matrix composition that is approximately 60–80% water based on the wet weight of the articular tissue. The high water content is important for the mechanical load-bearing capacity of cartilage tissue and, together with proteoglycans, for its "sponge effect." Natural articular cartilage also contains structural matrix macromolecules, such as collagen, proteoglycans, and non-collagenous proteins, in addition to water. Here, structural macromolecules account for approximately 20–40% of the wet weight of articular cartilage tissue.

[0006] It is already known to culture human chondrocytes three-dimensionally on agarose substrates, mimicking certain processes of embryonic development to produce cell aggregates with superior differentiation potential compared to monolayer cells, e.g., cartilage-like properties. These properties, which mirror native articular cartilage tissue as closely as possible, are characterized by the expression of collagen II (the main structural protein in the extracellular matrix of hyaline cartilage), proteoglycans such as aggrecan, and the intracellular chondrocyte-specific protein S100. Furthermore, it is desirable that the expression of type I collagen, which is inevitably upregulated during the cell proliferation phase of monolayer cultures, be reduced again in the cell aggregates, since this protein is almost absent in native articular cartilage. Therefore, since 2002, the present applicant (co.don AG) has proposed cell aggregates (spheroids) created in this way, primarily for the treatment of smaller cartilage defects caused by trauma. For this purpose, native articular cartilage tissue is harvested from patients, and cells isolated from it are propagated and cultured in 3D before transplantation as spheroids. The autologous chondrocyte implantation (ACI) product is known as Spherox®.

[0007] The culture of human cells in three-dimensional cell aggregates, particularly in a form known as spheroids, has already been approved for clinical use in humans, for example as autologous cartilage grafts (see Patent Document 1). Patent Document 1 relates to an in vitro method for producing three-dimensional cartilage and bone tissue from osteoblasts, chondrocyte stem cells, or mesenchymal stem cells. In this case, cells are first cultured in a monolayer and then in suspension until cell aggregates containing at least 40% by volume of extracellular matrix in which differentiated cells are embedded are produced. In this method, cell aggregates are produced by culturing cells in an agarose-coated cell culture vessel for at least 1 to 2 weeks.

[0008] Patent Document 2 discloses an in vitro method for producing three-dimensional cartilage tissue and bone tissue, 5 The patent discloses a method for producing spheroids from osteoblasts, chondrocytes, or mesenchymal stem cells by culturing 1×10 cells for at least two weeks. The spheroids produced according to Patent Document 2 have a diameter of 500 to 700 μm after one week. According to this method, 1×10 cells are cultured to produce spheroids. 5 pcs or 2 x 10 5 Chondrocytes are cultured for 5 days, 2 weeks, 1, 2 and 3 months.

[0009] According to this technique, a tissue biopsy from a patient is used as the starting material, and tissue-engineering cells are isolated from the biopsy by conventional methods, such as enzymatic digestion of the tissue, migration, or using reagents that recognize target cells.

[0010] These cells are then cultured in static culture, first in a substrate-supported monolayer (2D) in standard medium in a cell culture vessel with a particularly hydrophobic surface, and then in suspension by a simple substrate-free method until they form 3D cell aggregates containing at least 40% by volume and up to 95% by volume of extracellular matrix (ECM) with differentiated cells embedded within. These aggregates are called spheroids.

[0011] As a cell culture vessel, the culture process in suspension requires the use of a cell culture vessel with a hydrophobic, i.e., adhesion-preventing, surface such as polystyrene or Teflon®. Cell culture vessels with non-hydrophobic surfaces can be made hydrophobic, preferably by coating with agar or agarose. No further additives are required. Preferably, a 96-well plate is used as the cell culture vessel.

[0012] It is surprising that cells embedded in spheroids prepared using this technology survive and that the internal cells do not die even after long-term culture. With increasing culture time, the cells within these aggregates differentiate to form spheroids composed of ECM-differentiated cells and a surrounding proliferation zone. The process of tissue-specific matrix formation by embedded cells closely resembles the process of tissue formation, or regeneration and reconstruction, in vivo. During differentiation of cell cultures, the spacing between the aggregated cells increases due to the formation of the tissue-specific matrix. A tissue structure closely resembling that of natural tissue develops within the spheroids. Similar to natural cartilage, cells within the spheroids receive nutrients only through diffusion. During the further process of spheroid preparation, a cell zone capable of proliferation and migration forms at the spheroid's borders. This zone is highly advantageous in that, after the spheroid is incorporated into the defect, cells located in this peripheral zone can migrate and actively contact the surrounding tissue, and / or allow the in vitro-prepared tissue to integrate into its environment. Therefore, the tissue-specific cell aggregates produced are highly suitable for use in the treatment of tissue defects and in the in vitro and in vivo regeneration of tissue. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] German Patent Application Publication No. 10013223 A1 [Patent Document 2] US Patent No. 7887843 B2

Patent document 3

Patent document 4

Non-licensed literature

[0014]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

[0015] The aim of the present invention is to provide an improvement over known methods and hitherto available spheroids, in particular to reduce the outcome of non-response or treatment failure in a patient or patient population, and to provide a method for greater clinical safety for patients. [Means for solving the problem]

[0016] Such non-responders can be identified by an 8-10 point change in the so-called KOOS score (Knee injury and Osteoarthritis Outcome Score) compared to the preoperative state (see Non-Patent Document 1).

[0017] Surprisingly, the inventors concluded from studies including 120 patients (NCT01225575 and NCT0122259) that the operating width or process parameters associated with the method significantly influence the efficacy of spheroids.

[0018] Therefore, the underlying technical problem is solved by at least one requirement in which the incubation time is a key feature.

[0019] In particular, the present invention provides a method for proliferating or increasing chondrocytes and providing spheroids thereof, comprising the steps of: a) isolating chondrocytes from tissue of human or animal origin; b) culturing the cells from step a) in a spread monolayer (2D), wherein the monolayer is disposed on a substrate; c) harvesting the monolayer from step a) and culturing said cells in suspension in a 3D environment; d) wherein the total culture time of step b) and step c) is 55 days or less calculated from the start time of step a), and the time span of step b) is more than 10 days; e) selecting the resulting spheroids; This relates to a method, including

[0020] In a preferred embodiment of the invention, the time span of step b) is less than 38 days and / or the time span of step c) is less than 28 days.

[0021] In a preferred embodiment of the present invention, the time span of step b) is more than 10 days, or more than 16 days, or more than 20 days, and less than 38 days.

[0022] In a preferred embodiment of the present invention, the time span of step b) is more than 10 days and not more than 16 days.

[0023] Surprisingly, the resulting spheroids are of optimized quantity and size (diameter), respectively, thus providing spheroids of the best efficacy resulting in a sustainable reduction in non-responding or treatment-failed patients or patient populations, particularly those with a KOOS value of less than 8 as outlined below.

[0024] Therefore, the resulting and selected spheroids consist of cells in an optimized range of 3,000 to 200,000 cells, preferably in the range of 3,500 to 75,000 cells.

[0025] Furthermore, the diameter (size) of the spheroids is in an optimized range of 100 to 1,000 μm, preferably 200 to 900 μm, or preferably 240 to 870 μm.

[0026] Such spheroids obtained or selected were cultured in a 10 cm 2 It is most effective in treating and potentially curing defects, in which case 10-70 spheroids / cm 2 is used.

[0027] The spheroids thus obtained are further processed to prepare a pharmaceutical product.

[0028] In the sense of the present invention, "substrate" means any material suitable for supporting a monolayer.

[0029] The culture is carried out using cell culture conditions and media known to those skilled in the art (see Non-Patent Document 2).

[0030] The methods of the present invention allow for the beneficial expansion and proliferation of chondrocytes and spheroids thereof.

[0031] According to the present invention, a negative correlation was observed between the KOOS value and process parameters such as the monolayer culture time before the first passage (abbreviated as P0) (Spearman, P = 0.025) (Figure 1A), the culture time of spheroids (abbreviated as 3D) (P = 0.026) (Figure 1B), and the total culture time of chondrocytes in monolayer (abbreviated as ML) and spheroids (P = 0.007) (Figure 1C).

[0032] In Figure 1, the KOOS overall delta score is plotted against cell culture time in P0, 3D, and ML+3D for all spheroid batches from a patient, demonstrating a negative correlation (Figures 1A-1C). These initial findings suggest that shorter culture times are beneficial for product quality, as reflected by better clinical outcomes after transplantation.

[0033] This is supported by the observation that the non-responder group (KOOS < 8) received spheroid batches manufactured with significantly longer culture times at P0 compared to the responder group (Figure 5A). Similarly, the total cell culture time for ML and 3D was longer on average in the non-responder group (Figure 5B). This highlights the need to establish and limit new operational ranges or process parameters for cell culture time based on research data to prevent adverse effects on spheroid efficacy and patient outcomes.

[0034] To determine the provisional maximum time for chondrocyte culture, batches with the longest culture times were removed from the dataset until no negative correlation was observed (Spearman's correlation coefficient). For ML culture time before the first passage (P0), the maximum culture time without adverse effects on clinical outcomes was set at 19 days. However, because the batch belonged to a non-responder on day 19 of culture at P0, the provisional maximum culture time was set at 18 days (Figure 1D). The provisional maximum allowable time for spheroid culture was set at 31 days (Figure 1E). When batches cultured for more than 16 days at P0 and batches cultured for more than 28 days in 3D were removed from the dataset, only batches with a total culture time of up to 55 days in ML and 3D remained. Surprisingly, no correlation was observed between total culture time and efficacy in this patient / batch group (Figure 1F).

[0035] Because the correlation coefficients between clinical outcomes and P0, 3D, and total culture time were very low (-0.2), we further analyzed the incidence of non-responders (KOOS score <8) for batches manufactured with longer culture times. This revealed a surprisingly high non-response rate of 80% in patients treated with spheroid batches cultured at ML P0 between 19 and 28 days, and 61.5% for batches cultured between 16 and 28 days (see Table 3). The batch cultured up to 16 days showed a non-response rate of 28%, which is significantly lower than the 31.7% non-responder rate in the overall clinical trial population. Considering the non-response rate, combined with the feasibility of culture times during manufacturing, we set a tentative maximum culture time of 16 days, but did not further restrict it beyond 16 days.

[0036] A similar analysis was performed to set a provisional limit for spheroid culture time. After removing spheroid batches cultured for longer than 32 days from the dataset, the negative correlation disappeared (Figure 1E). However, because all spheroid batches cultured for 32 days belonged to non-responders (n = 4), a provisional limit of 31 days was set. Furthermore, patients treated with spheroids cultured in 3D for 32 to 42 days showed a relatively high non-response rate of 59% (n = 17) (see Table 4), highlighting the need to also limit 3D culture time. To further reduce the non-response rate, we considered the possibility of limiting the culture time to 28 days. Indeed, the 50% non-response rate between 29 and 42 days of culture suggests that further limiting the 3D culture time may further improve response rates. In line with this, the non-response rate was lower in the group in which culture was limited from 42 days to a maximum of 28 days (non-response rate of 31.7% vs. 26.6%, respectively (see Table 4)). Again, based on non-response rates as well as the feasibility of spheroid culture time, we limited spheroid culture time to a maximum of 28 days.

[0037] As expected from the separate evaluation of ML and 3D culture time, a negative correlation was also observed between total culture time and clinical outcome (Figure 1C). To clarify the additive effect of ML and 3D in the manufacturing process, we first eliminated their individual effects by removing batches cultured for more than 16 days in P0 and more than 28 days in 3D from the dataset, which contained 52% non-responders (see Table 5). The spheroid batch group remaining in this analysis was those cultured for 55 days or less. In this group, no correlation was observed between culture time and clinical outcome, revealing no additive effect of P0 and 3D when batches were cultured within the newly established culture time (Figure 1F). Therefore, further restrictions based on non-response rate only consider the effect of total culture time (ML + 3D) on clinical efficacy or safety. The batch group cultured for up to 55 days showed a non-response rate of 26.1%, a significant improvement compared to the overall batch group's 31.7%.

[0038] Therefore, according to the best mode of the present invention, the total culture time should be limited to 55 days or less.

[0039] In summary, these analyses revealed a high non-response rate of batches at long culture times, highlighting the need to adjust the operating window for chondrocyte culture times.

[0040] Given the high incidence of non-responders among patients treated with batches with long culture times, we compared the non-responder and responder groups for prolonged culture times outside the newly established limits. From this point on, only patients included in this study were further analyzed, as this study group could be compared with the microfracture (MFX) treatment group. In this study, 25% of the total patient group (n = 12) did not respond to ACI treatment 1 year after implantation. Focusing on this group, two of the 12 non-responders (16.7%) were cultured for longer than 16 days at P0, whereas only 8.5% of the responders (n = 3 of 36) were cultured for longer than 3D culture times. A more significant discrepancy was observed between non-responders and responders for prolonged 3D culture times. Seven of the 12 batches (58.3%) in the non-responder group were cultured in 3D for longer than 28 days, whereas only 16.6% of the responder batches (n = 8) were cultured for longer than 28 days. The overrepresentation of batches produced outside the newly restricted incubation times in the non-responder group (66.7%) compared to the responder group (22.2%) again highlights the need to adjust the incubation times. In all study groups, 67% of the batches were produced according to the stricter operating range (OR) (subgroup 1), and 33% of the batches were produced outside the newly established incubation limits (subgroup 2).

[0041] If the operating range or process parameters are not related to clinical efficacy, the distribution of batches that do not produce clinical improvement after ACI should be similar between batches with short and long culture times. Thus, these analyses represent the first correlation between KOOS and culture time and indicate that the manufacturing process may cause, at least in part, a reduction in the efficacy of chondrocyte spheroids used for ACI.

[0042] Because limiting cell culture time may improve the clinical efficacy of ACI treatment, we quantified the potential improvement of each study batch by comparing it with the comparator MFX. After establishing new limits for cell culture time in ML, 3D, and total culture time, we performed a retrospective evaluation of clinical data. Patients were divided into two groups. This division was based on the exclusion of batches from the study dataset established as described above. These subgroups consisted of spheroid batches manufactured within the newly established operating ranges for the process parameters P0, 3D, and ML+3D (subgroup 1) or spheroid batches for which at least one of the culture steps was outside these ranges (subgroup 2). The difference in culture time between the two subgroups is shown in Figure 2.

[0043] Here, we show that the culture times in monolayer (ML) and 3D, as well as the total culture time (ML + 3D), were significantly longer in batches from subgroup 2 (Figures 2B-2D). However, for P0, no statistically significant differences were observed between the two subgroups (Figure 2A). This is due to the fact that some of the batches with prolonged 3D culture times, and therefore assigned to subgroup 2, still had P0 culture times within the newly established range.

[0044] To identify differences in clinical outcomes, subgroup 1, consisting of 32 patients treated with chondrocyte spheroids produced with a limited culture time, was compared with the entire study group. This subgroup contained a high proportion of responders: 87.5% compared with 75% in the entire study group (Figure 3). In contrast, the subgroup of 16 patients treated with spheroid batches cultured outside the newly established operating window showed only a 50% positive clinical outcome 1 year after implantation (Figure 3).

[0045] To compare the clinical improvement in patients treated with spheroids produced by the tailored manufacturing process, a superiority analysis was performed between these batches and microfracture treatment (MFX). See Table 1 below.

[0046] [Table 1]

[0047] One year after implantation, clinical outcomes of the ACI product were shown to be non-inferior to microfracture (MFX) surgery (Figure 4). The difference between the mean KOOS (overall delta) of chondrocyte spheroid-based ACI compared with MFX was 5.75 points in favor of ACI treatment one year after implantation. A lower bound of -1.03 at the 95% confidence interval (CI 95%) indicated non-inferiority of ACI treatment compared with MFX (Figure 4). To address the possibility of improving KOOS-based clinical outcomes for subgroups manufactured within the newly established operating range, the mean difference between ACI subgroup 1 and MFX KOOS scores was calculated. This subgroup of spheroid batches represents Spherox®'s current manufacturing process, and a mean difference of 10.21 and a lower bound of 2.53 compared with MFX (CI 95%) indicated superiority over MFX (Figure 4). Therefore, retrospective elimination of certain batches with long culture times from the dataset would result in a higher mean KOOS score in this group.

[0048] The present invention further relates to implants, grafts, and functional replacement tissues obtained by the methods of the present invention. The implants, grafts, or functional replacement tissues produced in vitro or in vivo can be introduced into the recipient's surrounding natural tissue, preferably by injection. Cell division does not occur in the spheroids produced by the methods of the present invention, i.e., in the implants, grafts, or functional replacement tissues. When functional tissues are introduced into natural tissues, the spheroids then adhere (adhere) to the natural tissue and migrate (migrate) into the gaps, but do not divide. This ensures optimal supply / recovery without the risk of uncontrolled proliferation of functional replacement tissues. For this purpose, the resulting spheroids of the present invention produced in vitro are injected into diseased or degraded tissues. To this end, an injection needle or other suitable administration system must have at least the diameter of the spheroids. Suitable transfer and injection systems are disclosed in the applicant's U.S. Pat. Nos. 5,629,999 and 5,629,999.

[0049] The present invention also relates to preparations, in particular pharmaceutical or tissue preparations, drugs, grafts or implants, consisting of or comprising the spheroids obtainable by the method of the invention and, optionally, further excipients and additives.

[0050] The present invention also relates to pharmaceuticals, tissue preparations and drugs, in particular suspensions and solutions, especially injectable solutions, comprising the spheroids according to the invention and, optionally, further excipients and additives.

[0051] The present invention also relates to specific therapeutic / medicinal uses of the medicament according to the invention, the tissue preparation, the drug according to the invention, the graft according to the invention or the implant according to the invention for the treatment of cartilage and / or bone defects, in particular traumatic cartilage and / or bone defects, lesions, in particular traumatic lesions, cartilage degeneration, bone degeneration, osteoarthritis, and for cartilage regeneration therapy and / or bone regeneration in vivo. [Brief explanation of the drawings]

[0052] [Figure 1] 1 is a graph showing that clinical outcomes correlate with cell culture time. [Figure 2] 1 is a graph showing the chondrocyte culture time in subgroup 1 and subgroup 2 of this study. [Figure 3] FIG. 1 is a pie chart showing that spheroid batches with shorter culture times have improved response rates. [Figure 4] Graph showing superiority / non-inferiority analysis of subgroups 1 and 2. [Figure 5] Graph showing longer chondrocyte culture time in the group of non-responder patients. [Example]

[0053] The present invention will be described with reference to the following figures and examples, but the present invention is not limited to these figures and examples.

[0054] A total of 120 patients were included in the study (see above) for statistical correlation analysis between clinical outcomes and operating width or process parameters. The primary endpoint for evaluating clinical effectiveness used in this study was the change from baseline in the Knee Injury and Osteoarthritis Outcome Score (KOOS). Data from 72 patients, 48 ​​of whom underwent ACI and 49 of whom underwent MFX treatment, were evaluated in this study. These are shown in Table 2.

[0055] [Table 2]

[0056] The total KOOS score (including all subscales) was used before arthroscopy (baseline) and 1 year after implantation

[19] . Clinical outcome was defined as a positive overall change from baseline, a difference of at least 8 points between the two scores, based on a minimally important change (MIC) of 8–10, as described in the user's guide for the KOOS questionnaire (www.koos.nu).

[0057] The operating range of monolayer culture time, spheroid culture time, and total culture time between various cell culture passages used in the manufacturing process was statistically evaluated against the clinical outcomes of all batches manufactured for this study.

[0058] To gain insight into whether the newly established ranges and limits for cell culture time may have an effect on product efficacy, the 48 study patients were then divided into one group (subgroup 1) treated with batches manufactured with the newly established cell culture time and a second group (subgroup 2) treated with batches manufactured outside these limits. The mean KOOS overall delta scores for the full group, subgroup 1, and subgroup 2 were compared with the MFX group (superiority analysis). Patient characteristics for the two subgroups are shown in Table 2.

[0059] statistical analysis Differences between means were tested using the Mann-Whitney unpaired t-test (two-tailed t-test, P < 0.05 considered significant). Correlation analysis was performed using Spearman's correlation coefficient, with P < 0.05 considered significant. Superiority / non-inferiority analysis of ACI treatment compared with MFX treatment was performed using an unpaired t-test with Welch's correction at a 95% confidence interval. All statistical analyses were performed using GraphPad Prism v6 (GraphPad Software, USA).

[0060] Figure 1. Clinical outcomes correlate with cell culture time. From the 120 spheroid batches used in this study, we evaluated the operating window for clinical outcomes one year after transplantation. Longer culture times in monolayer (P0) (Figure 1A), 3D (spheroid) (Figure 1B), and total ML and 3D (Figure 1C) were correlated with lower KOOS overall delta scores. Removing spheroid batches from the dataset produced with longer culture times reduced the correlations for P0 (Figure 1D), 3D (Figure 1E), and total culture time (Figure 1F). This allowed us to tentatively limit the maximum culture time (arrows) below which the efficacy of the drug would not be adversely affected. The maximum culture time for P0 was set at 18 days (Figure 1D), for 3D at 31 days (Figure 1E), and for total ML and 3D culture time at 55 days (Figure 1F). r is Spearman correlation. A P value of <0.05 was considered significant. The KOOS is the Knee Injury and Osteoarthritis Outcome Score.

[0061] Figure 2. Chondrocyte culture time in subgroups 1 and 2 of this study The study batches were divided into two subgroups: Subgroup 1, cultured within the newly established maximum culture time, and Subgroup 2, cultured outside these limits. Considering the total culture time in ML (P = 0.003) and 3D (P < 0.001) (Figure 2C), and the total culture time in ML and 3D (P < 0.001) (Figure 2D), spheroid batches were cultured significantly longer in Subgroup 2. At P0 (Figure 2A), no significant difference in culture time was observed between the two subgroups (P = 0.075). This is because this subgroup includes batches with long 3D culture times (>28 days) that were cultured in OR (<16 days) at P0. The means between these groups were analyzed using a two-tailed Mann-Whitney t-test, with a P value < 0.05 considered significant.

[0062] Figure 3. Spheroid batches with shorter culture times show improved reaction rates Patients who underwent ACI were divided into two subgroups: subgroup 1 consisted of patients treated with spheroid batches manufactured with a limited culture time, and subgroup 2 consisted of patients treated with spheroid batches manufactured with a long culture time. Figure 3A: From the entire study group of patients, 75% of the spheroid batches belonged to responding patients (KOOS > 8). In subgroup 1 (Figure 3B), 87.5% of patients showed adequate clinical improvement (KOOS > 8), whereas in subgroup 2 (Figure 3C), only 50% of patients did so. Figure 3D: In the study control group (MFX), 69% of patients showed clinical improvement (KOOS > 8) after 1 year.

[0063] Figure 4. Superiority / non-inferiority analysis of subgroups 1 and 2 Clinical outcomes one year after transplantation for patients included in this study demonstrate noninferiority compared with microfracture (MFX). Two subgroups from this study containing spheroid batches were either cultured in a newly established OR (Subgroup 1) or cultured outside a newly established OR (Subgroup 2). One year after treatment, the mean KOOS global delta scores for the total patient group, Subgroup 1, and Subgroup 2 were compared with the mean KOOS global delta score for patients treated with MFX. Retrospective selection of spheroid batches produced within a more limited culture time resulted in the selection of patients with clinical outcomes demonstrating superiority over MFX treatment.

[0064] Figure 5. Longer chondrocyte culture time in the group of non-responders Of the 120 patients included in this study, non-responders (KOOS overall Δ score <8) received spheroid batches manufactured with significantly longer culture times at P0 (Figure 5A) and total culture time (Figure 5B). p<0.05 was used in the Mann-Whitney t-test. P0 is the monolayer culture time of isolated chondrocytes before the first passage.

[0065] Table 3: Setting the operating range (OR) for the process of the present invention using the non-response rate related to cell culture time. Patient IDs are shown in gray and marked with "N" correspond to non-responding patients (KOOS<8).

[0066] [Table 3-1]

[0067] [Table 3-2]

[0068] [Table 3-3]

[0069] [Table 3-4]

[0070] Table 4: Setting of the operating range (OR) for the process of the present invention using the non-response rate related to the culture time of spheroids. Patient IDs are shown in gray, and those marked with "N" correspond to non-responding patients (KOOS<8).

[0071] [Table 4-1]

[0072] [Table 4-2]

[0073] [Table 4-3]

[0074] Table 5: Setting of the operating range (OR) for the process of the present invention using the non-response rate related to the total culture time (monolayer and 3D). Patient IDs are shown in grey and marked with "N" correspond to non-responding patients (KOOS<8).

[0075] [Table 5-1]

[0076] [Table 5-2]

[0077] [Table 5-3]

Claims

1. A method for growing or increasing chondrocytes and providing spheroids thereof, comprising: a) providing chondrocytes isolated from tissue of human or animal origin as starting material; b) culturing the cells from step a) in a monolayer (2D) spread, wherein the monolayer is disposed on a substrate; c) harvesting the monolayer from step b) and culturing said cells in a 3D environment; d) wherein the total culture period of steps b) and c) is 55 days or less, calculated from the start time of step a), and the period range of step c) is 15 days or more and 28 days or less; e) selecting the resulting spheroids; A method comprising:

2. 2. The method of claim 1, wherein step b) is the total period during which the cells are cultured in a monolayer up to the first passage and after the first passage, and the monolayer culture period before the first passage is more than 10 days and 16 days or less.

3. 10. The method of claim 1, wherein the spheroids consist of more than 3,000 cells.

4. 2. The method of claim 1, wherein the spheroids consist of between 3,000 and 200,000 cells.

5. 5. The method of claim 4, wherein the spheroids consist of between 3,500 and 75,000 cells.

6. 2. The method of claim 1, wherein the spheroids have a diameter in the range of 100 to 1,000 μm.

7. 7. The method of claim 6, wherein the diameter of the spheroids is in the range of 200 to 900 μm or 240 to 870 μm.

8. A method according to any one of claims 1 to 7, wherein the obtained spheroids are for treating patients with cartilage and / or bone defects, wherein the treatment reduces the outcome of patients who are unresponsive or have treatment failure.

9. The method according to claim 8, wherein the obtained spheroids are for treating traumatic cartilage defects and / or bone defects and lesions.

10. The method according to claim 9, wherein the obtained spheroids are for the treatment of traumatic lesions, cartilage degeneration, bone degeneration, osteoarthritis, or for in vivo cartilage and / or bone regeneration therapy.

11. 11. The method of any one of claims 8 to 10, wherein the patient has a Knee Injury and Osteoarthritis Outcome Score (KOOS) of less than 8.

12. Spheroids obtainable by the method according to any one of claims 1 to 11.

13. A medicament comprising the spheroids of claim 12 for use in treating cartilage and / or bone defects.

14. A pharmaceutical comprising the spheroid of claim 13 for use in treating traumatic cartilage and / or bone defects and lesions.

15. 14. A pharmaceutical comprising the spheroids of claim 13 for use in the treatment of traumatic lesions, cartilage degeneration, bone degeneration, osteoarthritis, or for in vivo cartilage and / or bone regeneration therapy.

16. A pharmaceutical comprising the spheroid of claim 12.

17. Implants, grafts and functional replacement tissues comprising the spheroids of claim 12.

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